Contulakin-G, analogs thereof and uses thereof

ABSTRACT

The present invention is directed to contulakin-G (which is the native glycosylated peptide), a des-glycosylated contulakin-G (termed Thr 10 -contulakin-G), and derivatives thereof, to a cDNA clone encoding a precursor of this mature peptide and to a precursor peptide. The invention is further directed to the use of this peptide as a therapeutic for anti-seizure, anti-inflammatory, anti-shock, anti-thrombus, hypotensive, analgesia, anti-psychotic, Parkinson&#39;s disease, gastrointestinal disorders, depressive states, cognitive dysfunction, anxiety, tardive dyskinesia, drug dependency, panic attack, mania, irritable bowel syndrome, diarrhea, ulcer, GI tumors, Tourette&#39;s syndrome, Huntington&#39;s chorea, vascular leakage, anti-arteriosclerosis, vascular and vasodilation disorders, as well as neurological, neuropharmalogical and neuropsychopharmacological disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 09/420,797 filed on Oct. 19, 1999. The present application isrelated to U.S. provisional patent application Serial No. 60/105,015,filed on Oct. 20, 1998, Serial No. 60/128,561, filed on April 9, 1999and Serial No. 60/130,661, filed on Apr. 23, 1999, each incorporatedherein by reference, and claims priority to each under 35 USC §119(e).

This invention was made with Government support under Grant No. GM-48677awarded by the National Institutes of Health, Bethesda, Md. The UnitedStates Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

The present invention is directed to contulakin-G (which is the nativeglycosylated peptide), a des-glycosylated contulakin-G (termedThr₁₀-contulakin-G), and derivatives thereof, to a cDNA clone encoding aprecursor of this mature peptide and to a precursor peptide. Theinvention is further directed to the use of this peptide as atherapeutic for anti-seizure, anti-inflammatory, anti-shock,anti-thrombus, hypotensive, analgesia, anti-psychotic, Parkinson'sdisease, gastrointestinal disorders, depressive states, cognitivedysfunction, anxiety, tardive dyskinesia, drug dependency, panic attack,mania, irritable bowel syndrome, diarrhea, ulcer, GI tumors, Tourette'ssyndrome, Huntington's chorea, vascular leakage, anti-arteriosclerosis,vascular and vasodilation disorders, as well as neurological,neuropharmalogical and neuropsychopharmacological disorders.

The publications and other materials used herein to illuminate thebackground of the invention, and in particular, cases to provideadditional details respecting the practice, are incorporated byreference, and for convenience are numerically referenced in thefollowing text and respectively grouped in the appended bibliography.

Mollusks of the genus Conus produce a venom that enables them to carryout their unique predatory lifestyle. Prey are immobilized by the venomthat is injected by means of a highly specialized venom apparatus, adisposable hollow tooth that functions both in the manner of a harpoonand a hypodermic needle.

Few interactions between organisms are more striking than those betweena venomous animal and its envenomated victim. Venom may be used as aprimary weapon to capture prey or as a defense mechanism. Many of thesevenoms contain molecules directed to receptors and ion channels ofneuromuscular systems.

Several peptides isolated from Conus venoms have been characterized.These include the α-, μ- and ω-conotoxins which target nicotinicacetylcholine receptors, muscle sodium channels, and nouronal calciumchannels, respectively (Olivera et al., 1985). Conopressins, which arevasopressin analogs, have also been identified (Cruz et al.. 1987). Inaddition, peptides named conantokins have been isolated from Conusgeographus and Conus tulipa (Mena et al., 1990; Haack et al., 1990).These peptides have unusual age-dependent physiological effects: theyinduce a sleep-like state in mice younger than two weeks and hyperactivebehavior in mice older than 3 weeks (Haack et al., 1990). The isolation,structure and activity of K-conotoxins are described in U.S. Pat. No.5,633,347. Recently, peptides named contryphans containing D-tryptophanresidues have been isolated from Conus radiatus (U.S. Ser. No.09/061,026), and bromo-tryptophan conopeptides have been isolated fromConus imperialis and Conus radiatus (U.S. Ser. No. 08/785,534).

It is desired to identify additional conopeptides having activities ofthe above conopeptides, as well as conotoxin peptides having additionalactivities.

SUMMARY OF THE INVENTION

The present invention is directed to contulakin-G (which is the nativeglycosylated peptide), a des-glycosylated contulakin-G (termedThr₁₀-contulakin-G), and derivatives thereof, to a cDNA clone encoding aprecursor of this mature peptide and to a precursor peptide. Theinvention is further directed to the use of this peptide as atherapeutic for anti-seizure, anti-inflammatory, anti-shock,anti-thrombus, hypotensive, analgesia, anti-psychotic, Parkinson'sdisease, gastrointestinal disorders, depressive states, cognitivedysfunction, anxiety, tardive dyskinesia, drug dependency, panic attack,mania, irritable bowel syndrome, diarrhea, ulcer, GI tumors, Tourette'ssyndrome, Huntington's chorea, vascular leakage, anti-arteriosclerosis,vascular and vasodilation disorders, as well as neurological,neuropharmacological and neuropsychopharmacological disorders.

In one embodiment, the present invention is directed to contulakin-G,contulakin-G propeptide and nucleic acids encoding this peptide. Thecontulakin-G has the following formula:

Xaa₁-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Xaa₂-Tyr-Ile-Leu (SEQID NO:1)

where Xaa₁ is pyro-Glu, Xaa₂ is proline or hydroxyproline and Thr₁₀ ismodified to contain an O-glycan. X₂ is preferably proline. In accordancewith the present invention, a glycan shall mean any N-, S- or O-linkedmono-, di-, tri-, poly- or oligosaccharide that can be attached to anyhydroxy, amino or thiol group of natural or modified amino acids bysynthetic or enzymatic methodologies known in the art. Themonosaccharides making up the glycan can include D-allose, D-altrose,D-glucose, D-mannose, D-gulose, D-idose, D-galactose, D-talose,D-galactosamine, D-glucosamine, D-N-acetyl-glucosamine (GlcNAc),D-N-acetyl-galactosamine (GalNAc), D-fucose or D-arabinose. Thesesaccharides may be structurally modified as described herein, e.g., withone or more O-sulfate, O-phosphate, O-acetyl or acidic groups, such assialic acid, including combinations thereof. The gylcan may also includesimilar polyhydroxy groups, such as D-penicillamine 2,5 and halogenatedderivatives thereof or polypropylene glycol derivatives. The glycosidiclinkage is beta and 1-4 or 1-3, preferably 1-3. The linkage between theglycan and the amino acid may be alpha or beta, preferably alpha and is1-. Preferred glycans are described further herein, with the mostpreferred glycan being Gal(β1→3)GalNAc(α1→).

In a second embodiment, the present invention is directed to a genericcontulakin-G having the following general formula,

Xaa₁-Xaa₂-Xaa₃-Xaa₃-Gly-Gly-Xaa₂-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Ile-Leu(SEQ ID NO:2),

where Xaa₁ is pyro-Glu, Glu, Gln or γ-carboxy-Glu; Xaa₂ is Ser, Thr orS-glycan modified Cys; Xaa₃ is Glu or γ-carboxy-Glu; Xaa₄ is Asn,N-glycan modified Asn or S-glycan modified Cys; Xaa₅ is Ala or Gly; Xaa₆is Thr, Ser, S-glycan modified Cys, Tyr or any unnatural hydroxycontaining amino acid (such as 4-hydroxymethyl-Phe, 4-hydroxyphenyl-Gly,2,6-dimethyl-Tyr, 3-nitro-Tyr and 5-amino-Tyr); Xaa₇ is Lys,N-methyl-Lys, N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine,homoarginine or any unnatural basic amino acid (such asN-1-(2-pyrazolinyl)-Arg); Xaa₈ is Ala, Gly, Lys, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine, homoarginine, anyunnatural basic amino acid (such as N-1-(2-pyrazolinyl)-Arg) or X-Lyswhere X is (CH₂)_(n), phenyl, —(CH₂)_(m)—(CH═CH)—(CH₂)_(m)H or—(CH₂)_(m)—(C≡C)—(CH₂)_(m)H in which n is 1-4 and m is 0-2; Xaa₉ is Proor hydroxy-Pro; and Xaa₁₀ is Tyr, mono-iodo-Tyr, di-iodo-Tyr,O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, Trp, D-Trp, bromo-Trp,bromo-D-Trp, chloro-Trp, chloro-D-Trp, Phe, L-neo-Trp, any unnaturalaromatic amino acid (such as nitro-Phe, 4-substituted-Phe wherein thesubstituent is C₁-C₃ alkyl, carboxyl, hyrdroxymethyl, sulphomethyl,halo, phenyl, —CHO, —CN, —SO₃H and -NHAc, 2,6-dimethyl-Tyr and5-amino-Tyr). The C-terminus contains a free carboxyl group, is amidatedis acylated, contains a glycan or contains an aldehyde. It is preferredthat the C-terminus contains a free carboxyl. This peptide may furthercontain one or more glycans as described above. The glycans may occur atresidues 2, 7, 8, 10 and 16. The above and other unnatural basic aminoacids, unnatural hydroxy containing amino acids or unnatural aromaticamino acids are described in Building Block Index, Version 2.2,incorporated herein by reference, by and available from RSP Amino AcidAnalogues, Inc., Worcester, Mass.

In a third embodiment, the present invention is directed to analogs ofcontulakin-G or the generic contulakin-G. These analogs includeN-terminal truncations of contulakin-G or the generic contulakin-G up toand including Thr₁₀. When the N-terminal truncation is through Thr₁₀,Lys₁₁ is N-glycosylated using a carboxylated modified linker. ThisN-glycosylated Lys₁₁ can be represented as shown in FIG. 1 (Toth et al.,1999), in which R₂, R₃ and R₄ are as described herein. In thesetruncations, it is preferred that the residue proximal to the truncationis substituted with a glycosylated serine. Additional analogs includepeptides in which Ser-O-glycan, Thr-O-glycan or Cys-S-glycan issubstituted for a residue at position 1-9.

In a fourth embodiment, the present invention is directed to uses of thepeptides described herein as a therapeutic for anti-seizure,anti-inflammatory, anti-shock, anti-thrombus, hypotensive, analgesia,anti-psychotic, Parkinson's disease, gastrointestinal disorders,depressive states, cognitive dysfunction, anxiety, tardive dyskinesia,drug dependency, panic attack, mania, irritable bowel syndrome,diarrhea, ulcer, GI tumors, Tourette's syndrome, Huntington's chorea,vascular leakage, anti-arteriosclerosis, vascular and vasodilationdisorders, as well as neurological, neuropharmacological andneuropsychopharmacological disorders. In one aspect of this embodiment,analgesia is induced in a mammal using one of the peptides describedherein. In a second aspect of this embodiment, epilepsy or convulsionsare treated in a mammal. In a third aspect of this embodiment,schizophrenia is treated in an mammal. In a fourth aspect of thisembodiment, tardive dyskinesia and acute dystonic reactions are treatedin a mammal. In a fifth aspect of this embodiment, inflammation istreated in a mammal.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of an N-glycosylation of Lys using acarboxylated modified linker.

FIG. 2 shows the native O-glycan attached to Thr₁₀ of contulakin-G.

FIG. 3 shows analogs of the glycan which can be attached to one or moreresidues of contulakin-G.

FIG. 4 shows the preferred core O-glycans (Van de Steen et al., 1998).Mucin type O-linked oligosaccharides are attached to Ser or Thr (orother hydroxylated residues of the present peptides) by a GalNAcresidue. The monosaccharide building blocks and the linkage attached tothis first GalNAc residue define the “core glycans,” of which eight havebeen identified. The type of glycosidic linkage (orientation andconnectivities) are defined for each core glycan.

FIG. 5 shows the purification of Contulakin-G. One gram of crudelyophilized venom from Conus geographus was extracted and applied on aSephadex G-25 column as previously described (Olivera et al., 1984).Three successive fractions containing paralytic and sleeper activities(Ve/Vo=1.37 to 1.41) were pooled, applied on a preparative reversedphase Vydac C₁₈ column and eluted with a gradient of acetonitrile in0.1% trifluroacetic acid. The component indicated by an arrow in panel Acaused wobbling and death when administered icv in mice.

FIG. 6 shows a nano-ESI MS/MS spectrum (m/z 1035 precursor) of nativecontulakin-G (286-1886 Da) (the MS/MS experiment is denoted using asuggested shorthand (Mcluckey et al., 1991) where the closed circlerepresents m/z 1035 [M+2H]²⁺ precursor and the arrows are directedtowards the open circles which represent the fragments generated fromthe precursor). Above the spectrum, the structure of the glycoamino acidis represented where the arrows indicate 2 sites which lead to majorfragment ions observed in the MS/MS spectrum (Craig et al., 1993).

FIGS. 7A-7C show dose-response of CGX-1063 (Thr₁₀-contulakin-G) onspinally mediated (limb withdrawal) and supraspinally mediated (hindlimblick) nociceptive behaviors elicited by noxious heat. Data are expressedas seconds to response (FIGS. 7A and 7B) or to first fall (FIG. 7C). InFIG. 7A, latency to the first observable response after placement on a50° C. hotplate is shown. FIG. 7B shows latency to the first hindpawlick. FIG. 7C shows latency to first fall after placement on theaccelerating rotorod (in FIGS. 7A-7C, n=3-10).

FIGS. 8A-8B show the effect of CGX-1063 on the nociceptive response topersistent pain. In FIG. 8A, data are presented as the amount of timeanimals spent licking the formalin-injected hindpaw (n=7-10animals/treatment group). Intrathecal CGX-1063 dose-dependentlydecreased the phase 2 nociceptive response in the formalin test comparedto intrathecal saline injected controls. FIG. 8B shows the latency tofirst fall from an accelerated rotorod immediately following theformalin test.

FIG. 9 shows paw withdrawal threshold to mechanical stimulation one weekfollowing partial sciatic nerve ligation. Data are presented as the 50%withdrawal threshold in grams determined with calibrated von Freyfilaments (n=3-9 animals per group).

FIGS. 10A-10B show a comparison of CGX-1160 (contulakin-G), CGX-1063 andNT in the tail-flick test. Dose-response of the three compounds is shownin FIG. 10A. FIG. 10B shows the duration of effect at the highest dosestested for each compound (CGX-1160=100 pmol; CGX-1063=100 pmol; NT=10nmol).

FIGS. 11A-11B show the effect of CGX-1160, CGX-1063 and NT on phase 1(FIG. 11A) and phase 2 (FIG. 11B) of the formalin test. All three of thecompounds dose-dependently reduced nociceptive behavior following i.pl.formalin. In phase 2 (FIG. 11B), CGX-1160 was 10 times more potent thanCGX-1063, and 600-700 times more potent than NT.

FIGS. 12A-12C show effect of CGX-1160, CGX-1063 and NT on chronicinflammation-induced mechanical allodynia. Numbers in parenthesesindicate percentage of each corresponding control value. In FIG. 12A,CGX-1160 potently and dose-dependently reversed CFA-induced allodynia.In FIG. 12B, CGX-1063 reversed CFA-induced allodynia, but wasapproximately 100-fold less potent in this model than CGX-1160. In FIG.12C, NT reversed CFA-induced allodynia at 1,000 pmol, but not 100 pmol,approximately 10,000-fold less potent than CGX-1160.

FIGS. 13A-13B show locomotor impairing effects of CGX-1160, CGX-1063 andNT. FIG. 13A shows time to peak effect and duration of effect of thethree compounds at the highest doses tested (approximately 100 times theED₅₀ in phase 2 of the formalin test). FIG. 13B shows dose-response ofeach compound on locomotor impairment.

FIGS. 14A-14C show dose-effect and time to peak effect and duration oflocomotor impairment of CGX-1160, CGX-1063 and NT. FIG. 14A shows thatCGX-1160 caused long-lasting motor impairment only at doses 100-fold orgreater than its ED₅₀. FIG. 14B shows that CGX-1063 caused long-lastmotor impairment at doses 10-fold or greater than its ED₅₀. FIG. 14Cshows that NT caused long-last motor impairment at doses 100-foldgreater than its ED₅₀.

FIGS. 15A-15B show a comparison of CGX-1160, CGX-1063 and NT on changein body temperature. FIG. 15A shows time to peak effect and duration ofeach compound, and FIG. 15B shows dose-response of each compound.

FIGS. 16A-16C show hypothermic dose-effect and duration of CGX-1160,CGX-1063 and NT. In FIG. 16A, CGX-1160 caused hypothermia only at doses100-500 times greater than ED₅₀. FIG. 16B shows the long-lastinghypothermic effect of CGX-1063 at doses 10-fold higher than ED₅₀.(100pmol). In FIG. 16C, NT had a hypothalamic effect at doses 10-100 timeshigher than its ED₅₀.

FIG. 17 shows effects of Thr₁₀-g Contulakin-G (CGX-1160; 100 pmoli.c.v.) on D-amphetamine-stimulated locomotor activity as measured bydistance traveled. Abbreviations: sal-sal: i.p. treatment was saline,i.c.v. treatment was saline; amphet (3 mg/kg)-sal: i.p. treatment wasD-amphetamine sulphate (3 mg/kg), i.c.v. treatment was saline; amphet(10 mg/kg)-sal: i.p. treatment was D-amphetamine sulphate (10 mg/kg),i.c.v. treatment was saline; sal-ctl: i.p. treatment was saline, i.c.v.treatment was Thr₁₀-g contulakin-G (100 pmol); amphet (3 mg/kg)-ctl:i.p. treatment was D-amphetamine sulphate (3 mg/kg), i.c.v. treatmentwas Thr₁₀-g contulakin-G (100 pmol). Each bar shows the mean ±SEM of 3-7mice per group. a: P<0.05 vs saline-saline treated group (sal-sal); b:P<0.05 vs D-amphetamine-saline group (amphet (3 mg/kg)-sal).

FIG. 18 shows the effects of Thr₁₀-g Contulakin-G (CGX-1160; 100 pmoli.c.v.) on D-amphetamine-stimulated locomotor activity as measured bytime spent ambulatory (s). Abbreviations: sal-sal: i.p. treatment wassaline, i.c.v. treatment was saline; amphet (3 mg/kg)-sal: i.p.treatment was D-amphetamine sulphate (3 mg/kg), i.c.v. treatment wassaline; amphet (10 mg/kg)-sal: i.p. treatment was D-amphetamine sulphate(10 mg/kg), i.c.v. treatment was saline; sal-ctl: i.p. treatment wassaline, i.c.v. treatment was Thr₁₀-g contulakin-G (100 pmol); amphet (3mg/kg)-ctl: i.p. treatment was D-amphetamine sulphate (3 mg/kg), i.c.v.treatment was Thr₁₀-g contulakin-G (100 pmol). Each bar shows themean±SEM of 3∝7 mice per group. a: P<0.05 vs saline-saline treated group(sal-sal); b: P<0.05 vs D-amphetamine-saline group (amphet (3mg/kg)-sal).

FIG. 19 shows CGX-1160 and CGX-1063 dose-dependently protect againstaudiogenic seizures following i.c.v. administration in Frings mice, atdoses well below minimal motor impairing doses. Each point representsthe percent protection (toxic in groups of at least four mice).

FIG. 20 shows CGX-1160's long-lasting efficacy in blocking audiogenicseizures following i.c.v. administration in Frings mice. Neurotensin isonly 50% effective following i.c.v. administration of up to 5 nmol. Eachpoint represents the percent protection in a group of four mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to contulakin-G (which is the nativeglycosylated peptide), a des-glycosylated contulakin-G (termedThr₁₀-contulakin-G), and derivatives thereof, to a cDNA clone encoding aprecursor of this mature peptide and to a precursor peptide. Theinvention is further directed to the use of this peptide as atherapeutic for anti-seizure, anti-inflammatory, anti-shock,anti-thrombus, hypotensive, analgesia, anti-psychotic, Parkinson'sdisease, gastrointestinal disorders, depressive states, cognitivedysfunction, anxiety, tardive dyskinesia, drug dependency, panic attack,mania, irritable bowel syndrome, diarrhea, ulcer, GI tumors, Tourette'ssyndrome, Huntington's chorea, vascular leakage, anti-arteriosclerosis,vascular and vasodilation disorders, as well as neurological,neuropharmalogical and neuropsychopharmacological disorders.

The present invention is directed to contulakin-G and contulakin-Ganalogues as described above. These peptides may contain single ormultiple glycan post-translational modifications at one or more, up toall, of the hydroxyl sites of the peptides. The glycans are as describedherein. The native O-glycan attached to contulakin-G is shown in FIG. 2.FIG. 3 shows analogs of the glycan which can be attached to one or moreresidues of contulakin-G. In this figure, R₁ is an amino capable ofbeing derivatized with a gylcan either chemically or enzymatically; R₂is OH, NH₂, NHSO₃Na, NHAc, O-sulfate, O-phosphate, or O-glycan; R₃ is H,SO₃ , PO₃, acetyl, sialic acid monosaccharide; R₄ is H, SO₃, PO₃, acetylor monosaccharide; R₅ is OH, NH₂, NHSO₃Na, NHAc, O-sulfate, O-phosphate,O-monosaccharide or, O-acetyl; R₆ is H, SO₃, PO₃, acetyl ormonosaccharide; R₇ is H, SO₃, PO₃, acetyl or monosaccharide; R₈ is H,SO₃, PO₃, acetyl or monosaccharide; n is 0-4 and m is 1-4.

The preferred core glycans which can be used to modify contulakin-G oranalogs disclosed herein are shown in FIG. 4. Further branching fromthese cores using the monosaccharides described herein may also be made.Preferred glycosidic linkages are specified by cores 5 and 7 of FIG. 4with further homolgation of the glycan at positions 3, 4 and 6 of theGalNAc template using the monosaccharides described herein Any freehydroxy function may be O-sulfated, O-phosphorylated or O-aceylated.

The glycosylated conopeptide (contulakin-G or CGX-1160) has higher invivo potency than the unglycosylated conopeptide (Thr₁₀-contulakin-G orCGX-1063), although their in vitro potencies are about the same. Theglycosylation may be important for better binding with the receptor,and/or enhanced delivery of the conopeptide to its site of action,and/or inhibition of degradation of the conopeptide.

The present invention is further directed to DNA sequence coding forcontulakin-G as described in further herein. The invention is furtherdirected to the propeptide for contulakin-G as described in furtherdetail herein.

The present invention relates to a novel linear glycosylatedcontulakin-G, and derivatives thereof that are useful as pharmaceuticalagents, to methods for their production, to pharmaceutical compositionswhich include these compounds and a pharmaceutically acceptable carrier,and to pharmaceutical methods of treatment. The novel compounds of thepresent invention are central nervous system agents and their biologicalactions are effected at a novel “Contulakin-G binding site on theneurotensin receptor”. More particularly, the novel compounds of thepresent invention are analgesics, anti-inflammatory agents,antipsychotic agents for treating psychoses such as schizophrenia anddisplay potent anti-seizure properties in established animal models ofepilepsy.

PAIN: Chronic or intractable pain, such as may occur in conditions suchas bone degenerative diseases and cancer, is a debilitating conditionwhich is treated with a variety of analgesic agents, and often opioidcompounds, such as morphine.

In general, brain pathways governing the perception of pain are stillincompletely understood, sensory afferent synaptic connections to thespinal cord, termed “nociceptive pathways” have been documented in somedetail. In the first leg of such pathways, C- and A-fibers which projectfrom peripheral sites to the spinal cord carry nociceptive signals.Polysynaptic junctions in the dorsal horn of the spinal cord areinvolved in the relay and modulation of sensations of pain to variousregions of the brain, including the periaqueductal grey region.Analgesia, or the reduction of pain perception, can be effected directlyby decreasing transmission along such nociceptive pathways. Analgesicopiates are thought to act by mimicking the effects of endorphin orenkephalin peptide-containing neurons, which synapse presynaptically atthe C- or A- fiber terminal and which, when they fire, inhibit releaseof neurotransmitters, including substance P. Descending pathways fromthe brain are also inhibitory on C- and A-fiber firing.

Certain types of pain have complex etiologies. For example, neuropathicpain is generally a chronic condition attributable to injury or partialtransection of a peripheral nerve. This type of pain is characterized byhyperesthesia, or enhanced sensitivity to external noxious stimuli. Thehyperesthetic component of neuropathic pain does not respond to the samepharmaceutical interventions as does more generalized and acute forms ofpain.

Opioid compounds such as morphine, while effective in producinganalgesia for many types of pain, are not always effective, and mayinduce tolerance in patients. When a subject is tolerant to opioidnarcotics, increased doses are required to achieve a satisfactoryanalgesic effect. These compounds can produce side effects, such asrespiratory depression, which can be life threatening. In addition,opioids frequently produce physical dependence in patients. Dependenceappears to be related to the dose of opioid taken and the period of timeover which it is taken by the subject. For this reason, alternatetherapies for the management of chronic pain are widely sought after. Inaddition, compounds which serve as either a replacement for or as anadjunct to opioid treatment in order to decrease the dosage of analgesiccompound required, have utility in the treatment of pain, particularlypain of the chronic, intractable type.

Since contulakin-G has been shown to act at a site on certainneurotensin receptors, and neurotensin has been shown to have analgesicactions (Clineschmidt et al. 1979), then contulakin-G like conopeptidesare useful for the treatment of pain and related disorders.

SCHIZOPHRENIA: Schizophrenia is a neurogenic disorder that is currentlytreated primarily with neuroleptic compounds such as phenothiazines andbutyrophenones, which block dopamine receptors. Since contulakin-G hasbeen shown to act at a site on certain neurotensin receptors, andneurotensin actions are implicated in the etiology of schizophrenia(Nemeroff et al. 1992), then contulakin-G like conopeptides are usefulfor the treatment of schizophrenia and related disorders.

The in vitro selection criteria for conopeptides useful in treatingschizophrenia, include: a)activation of Contulakin-G sites; b) highaffinity reversible binding to a Contulakin-G binding site localized tothe limbic region of the brain, and c) inhibition of dopamine releasefrom brain regions, particularly limbic brain regions.

Compounds exhibiting sufficiently high activities in the above in vitroscreening assays are then tested in an animal model used in screeninganti-psychotic compounds.

TARDIVE DYSKINESIA AND OTHER ACUTE DYSTONIC REACTIONS: Tardivedyskinesia and acute dystonic reactions are movement disorders that arecommonly produced as side effects of anti-psychotic therapy employingdopamine antagonists, such as haloperidol. These disorders arecharacterized by supersensitivity of dopamine receptors in certainregions of the brain associated with control of movement, particularlythe basal ganglia. Currently, intermittent antipsychotic therapy is usedin attempt to avoid onset of the disorder, and such disorders aretreated by withdrawal of therapy.

Criteria for selection of an omega-conopeptide for treatment of tardivedyskinesia include: a) activation of Contulakin-G sites; b) highaffinity reversible binding to the Contulakin-G site ; c) inhibition ofdopamine release from striatal brain regions, and other regions of thebasal ganglia, and d) a ratio of inhibition of dopamine release in thebasal ganglia to inhibition of dopamine release in the limbic regions.

Compounds showing sufficiently high activities in in vitro screeningassays are then tested in the rat striatal turning model, describedabove. Compounds useful in the method of treating such movementdisorders, when injected to the striatum on the side of the braincontralateral to the lesion, correct the turning behavior.

INFLAMMATION: A neurogenic component of inflammation has been described,in that blockade of the sympathetic nervous system, and particularlyblockade of beta-adrenergic receptors, is helpful in reducinginflammatory joint damage. Compounds useful in the treatment ofinflammation would be expected to have the following in vitroproperties: a) activation of novel Contulakin-G sites; b) high affinitybinding to the Contulakin-G binding sites, and c) inhibition ofnorepinephrine release from nervous tissue. Compounds exhibitingsufficiently high activities in such in vitro screening assays aretested in an animal model of rheumatoid arthritis.

EPILEPSY: Epilepsy is a general term which describes disorders of thecentral nervous system characterized by repeated episodes of seizures.Such seizures may involve the sensory, autonomic or motor nervoussystems and are recognized electrophysiologically by the presence ofabnormal electric discharges in the brain. The pathophysiology of suchabnormal discharge activity is not well understood; however, there isevidence that loss of inhibitory neural input, such as GABA input, isinvolved in at least some epileptic seizures.

The ability of certain of the benzodiazepines (e.g., diazepam) torepress or inhibit epileptic episodes is considered by some to beevidence of a GABAergic pathophysiology in seizure activity, since thesedrugs are known to potentiate GABAergic neural inhibition via an effecton the GABA receptor-associated chloride ion channel. Biochemicaleffects of other anti-epileptic compounds include stabilization ofexcitable membranes by inhibition of voltage-sensitive sodium orpotassium channels (phenytoin), and general depression of neuronalfunction characterized by facilitation of GABAergic transmission,inhibition of the effects of excitatory (glutaminergic)neurotransmission and depression of neurotransmitter release(phenobarbital).

Compounds useful in the treatment of epilepsy would be expected to havethe following in vitro properties: a)activation of novel Contulakin-Gsites; b) high affinity binding to the contulakin-G conopeptide bindingsites, and c) inhibition of excitatory neurotransmitter release fromnervous tissue. Compounds exhibiting sufficiently high activities insuch in vitro screening assays fare tested in an established animalmodel of epilepsy.

In addition to the above specific disorders, since the peptides,derivatives and analogs of the present invention have been found to bindto the neurotensin receptor, these compounds are also useful inconnection with conditions associated with the neurotensin receptor andfor which neurotensin-like compounds or other compounds have been shownto be active. These activities include: methamphetamine antagonists,antipsychotic agents, cerebral medicaments, analgesic agents,anti-endotoxin shock effect, protease inhibition action (ananti-thrombin action, an anti-plasmin action), a hypotensive action, ananti-DIC action, an anti-allergic action, a wound healing action,cerebral edema, an edema of the lung, an edema of the trachea, athrombus, an arteriosclerosis, a burn, and a hypertension, allergicdiseases (such as a bronchial asthma and a pollenosis), reducinghemorrhage from a sharp trauma such as an injured tissue portion at thetime of surgical operation, a lacerated wound of a brain or othertissues caused by a traffic accident and the like, and for relaxing andcuring swelling, pain inflammation caused by trauma, suppressinginternal hemorrhage caused by a dull trauma, edemata and inflammationwhich are accompanied with the internal hemorrhage, suppression andimprovement of cerebral edemata by suppressing a leakage of bloodcomponents to a tissue matrix found in cerebral ischemetic diseaseswhich include cerebral infractions (e.g., a cerebral thrombus and acerebral embolism), intracranial hemorrhages (e.g., a cerebralhemorrhage and a subarachnoidal hemorrhage), a transient cerebralischemic attack, acute cerebral blood vessel disorders in a hypertensiveencephalopathy, suppression and improvement of burns, chilblains, otherskin inflammations and swelling, an upper tracheal inflammation, anasthma, nasal congestion, a pulmonary edema, and inflammable disorderscaused by endogenous and exogenous factors, which directly damagevascular endothelia and mucous membranes, such as an environmentalchemical substance, chemotherapeutics of cancer, an endotoxin, and aninflammation mediator.

The conopeptides of the present invention are identified by isolationfrom Conus venom. Alternatively, the conopeptides of the presentinvention are identified using recombinant DNA techniques by screeningcDNA libraries of various Conus species using conventional techniqueswith degenerate probes. Clones which hybridize to these probes areanalyzed to identify those which meet minimal size requirements, i.e.,clones having approximately 300 nucleotides (for a propeptide), asdetermined using PCR primers which flank the cDNA cloning sites for thespecific cDNA library being examined. These minimal-sized clones arethen sequenced. The sequences are then examined for the presence of apeptide having the characteristics noted above for conopeptides. Thebiological activity of the peptides identified by this method is testedas described herein, in U.S. Pat. No. 5,635,347 or conventionally in theart.

These peptides are sufficiently small to be chemically synthesized.General chemical syntheses for preparing the foregoing conopeptides aredescribed hereinafter, along with specific chemical synthesis ofconopeptides and indications of biological activities of these syntheticproducts. Various ones of these conopeptides can also be obtained byisolation and purification from specific Conus species using thetechniques described in U.S. Pat. No. 4,447,356 (Olivera et al., 1984),U.S. Pat. No. 5,514,774 (Olivera et al., 1996) and U.S. Pat. No.5,591,821 (Olivera et al., 1997), the disclosures of which areincorporated herein by reference.

Although the conopeptides of the present invention can be obtained bypurification from cone snails, because the amounts of conopeptidesobtainable from individual snails are very small, the desiredsubstantially pure conopeptides are best practically obtained incommercially valuable amounts by chemical synthesis using solid-phasestrategy. For example, the yield from a single cone snail may be about10 micrograms or less of conopeptide. By “substantially pure” is meantthat the peptide is present in the substantial absence of otherbiological molecules of the same type; it is preferably present in anamount of at least about 85% purity and preferably at least about 95%purity. Chemical synthesis of biologically active conopeptides dependsof course upon correct determination of the amino acid sequence. Thus,the conopeptides of the present invention may be isolated, synthesizedand/or substantially pure.

The conopeptides can also be produced by recombinant DNA techniques wellknown in the art. Such techniques are described by Sambrook et al.(1989). The peptides produced in this manner are isolated, reduced ifnecessary, and oxidized to form the correct disulfide bonds, if presentin the final molecule.

One method of forming disulfide bonds in the conopeptides of the presentinvention is the air oxidation of the linear peptides for prolongedperiods under cold room temperatures or at room temperature. Thisprocedure results in the creation of a substantial amount of thebioactive, disulfide-linked peptides. The oxidized peptides arefractionated using reverse-phase high performance liquid chromatography(HPLC) or the like, to separate peptides having different linkedconfigurations. Thereafter, either by comparing these fractions with theelution of the native material or by using a simple assay, theparticular fraction having the correct linkage for maximum biologicalpotency is easily determined. It is also found that the linear peptide,or the oxidized product having more than one fraction, can sometimes beused for in vivo administration because the cross-linking and/orrearrangement which occurs in vivo has been found to create thebiologically potent conopeptide molecule. However, because of thedilution resulting from the presence of other fractions of lessbiopotency, a somewhat higher dosage may be required.

The peptides are synthesized by a suitable method, such as byexclusively solid-phase techniques, by partial solid-phase techniques,by fragment condensation or by classical solution couplings.

In conventional solution phase peptide synthesis, the peptide chain canbe prepared by a series of coupling reactions in which constituent aminoacids are added to the growing peptide chain in the desired sequence.Use of various coupling reagents, e.g., dicyclohexylcarbodiimide ordiisopropyl-carbonyldimidazole, various active esters, e.g., esters ofN-hydroxyphthalimide or N-hydroxy-succinimide, and the various cleavagereagents, to carry out reaction in solution, with subsequent isolationand purification of intermediates, is well known classical peptidemethodology. Classical solution synthesis is described in detail in thetreatise, “Methoden der Organischen Chemie (Houben-Weyl): Synthese vonPeptiden,” (1974). Techniques of exclusively solid-phase synthesis areset forth in the textbook, “Solid-Phase Peptide Synthesis,” (Stewart andYoung, 1969), and are exemplified by the disclosure of U.S. Pat. No.4,105,603 (Vale et al., 1978). The fragment condensation method ofsynthesis is exemplified in U.S. Pat. No. 3,972,859 (1976). Otheravailable syntheses are exemplified by U.S. Pat. No. 3,842,067 (1974)and U.S. Pat. No. 3,862,925 (1975). The synthesis of peptides containingy-carboxyglutamic acid residues is exemplified by Rivier et al. (1987),Nishiuchi et al. (1993) and Zhou et al. (1996). Synthesis ofconopeptides have been described in U.S. Pat. No. 4,447,356 (Olivera etal., 1984), U.S. Pat. No. 5,514,774 (Olivera et al., 1996) and U.S. Pat.No. 5,591,821 (Olivera et al., 1997).

Common to such chemical syntheses is the protection of the labile sidechain groups of the various amino acid moieties with suitable protectinggroups which will prevent a chemical reaction from occurring at thatsite until the group is ultimately removed. Usually also common is theprotection of an α-amino group on an amino acid or a fragment while thatentity reacts at the carboxyl group, followed by the selective removalof the α-amino protecting group to allow subsequent reaction to takeplace at that location. Accordingly, it is common that, as a step insuch a synthesis, an intermediate compound is produced which includeseach of the amino acid residues located in its desired sequence in thepeptide chain with appropriate side-chain protecting groups linked tovarious ones of the residues having labile side chains.

As far as the selection of a side chain amino protecting group isconcerned, generally one is chosen which is not removed duringdeprotection of the α-amino groups during the synthesis. However, forsome amino acids, e.g., His, protection is not generally necessary. Inselecting a particular side chain protecting group to be used in thesynthesis of the peptides, the following general rules are followed: (a)the protecting group preferably retains its protecting properties and isnot split off under coupling conditions, (b) the protecting group shouldbe stable under the reaction conditions selected for removing theα-amino protecting group at each step of the synthesis, and (c) the sidechain protecting group must be removable, upon the completion of thesynthesis containing the desired amino acid sequence, under reactionconditions that will not undesirably alter the peptide chain.

It should be possible to prepare many, or even all, of these peptidesusing recombinant DNA technology. However, when peptides are not soprepared, they are preferably prepared using the Merrifield solid-phasesynthesis, although other equivalent chemical syntheses known in the artcan also be used as previously mentioned. Solid-phase synthesis iscommenced from the C-terminus of the peptide by coupling a protectedα-amino acid to a suitable resin. Such a starting material can beprepared by attaching an α-amino-protected amino acid by an esterlinkage to a chloromethylated resin or a hydroxymethyl resin, or by anamide bond to a benzhydrylamine (BHA) resin orpara-methylbenzhydrylamine (MBHA) resin. Preparation of thehydroxymethyl resin is described by Bodansky et al. (1966).Chloromethylated resins are commercially available from Bio RadLaboratories (Richmond, Calif.) and from Lab. Systems, Inc. Thepreparation of such a resin is described by Stewart and Young (1969).BHA and MBHA resin supports are commercially available, and aregenerally used when the desired polypeptide being synthesized has anunsubstituted amide at the C-terminus. Thus, solid resin supports may beany of those known in the art, such as one having the formulae—O—CH₂-resin support, —NH BHA resin support, or —NH-MBHA resin support.When the unsubstituted amide is desired, use of a BHA or MBHA resin ispreferred, because cleavage directly gives the amide. In case theN-methyl amide is desired, it can be generated from an N-methyl BHAresin. Should other substituted amides be desired, the teaching of U.S.Pat. No. 4,569,967 (Kornreich et al., 1986) can be used, or should stillother groups than the free acid be desired at the C-terminus, it may bepreferable to synthesize the peptide using classical methods as setforth in the Houben-Weyl text (1974).

The C-terminal amino acid, protected by Boc or Fmoc and by a side-chainprotecting group, if appropriate, can be first coupled to achloromethylated resin according to the procedure set forth in Horiki etal. (1978), using KF in DMF at about 60° C. for 24 hours with stirring,when a peptide having free acid at the C-terminus is to be synthesized.Following the coupling of the BOC-protected amino acid to the resinsupport, the α-amino protecting group is removed, as by usingtrifluoroacetic acid (TFA) in methylene chloride or TFA alone. Thedeprotection is carried out at a temperature between about 0° C. androom temperature. Other standard cleaving reagents, such as HCl indioxane, and conditions for removal of specific α-amino protectinggroups may be used as described in Schroder and Lubke (1965).

After removal of the α-amino-protecting group, the remaining α-amino-and side chain-protected amino acids are coupled step-wise in thedesired order to obtain the intermediate compound defined hereinbefore,or as an alternative to adding each amino acid separately in thesynthesis, some of them may be coupled to one another prior to additionto the solid phase reactor. Selection of an appropriate coupling reagentis within the skill of the art. Particularly suitable as a couplingreagent is N,N′-dicyclohexylcarbodiimide (DCC, DIC, HBTU, HATU, TBTU inthe presence of HoBt or HoAt).

The activating reagents used in the solid phase synthesis of thepeptides are well known in the peptide art. Examples of suitableactivating reagents are carbodiimides, such asN,N′-diisopropylcarbodiimide andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. Other activatingreagents and their use in peptide coupling are described by Schroder andLubke (1965) and Kapoor (1970).

Each protected amino acid or amino acid sequence is introduced into thesolid-phase reactor in about a twofold or more excess, and the couplingmay be carried out in a medium of dimethylformamide (DMF):CH₂Cl₂ (1:1)or in DMF or CH₂Cl₂ alone. In cases where intermediate coupling occurs,the coupling procedure is repeated before removal of the α-aminoprotecting group prior to the coupling of the next amino acid. Thesuccess of the coupling reaction at each stage of the synthesis, ifperformed manually, is preferably monitored by the ninhydrin reaction,as described by Kaiser et al. (1970). Coupling reactions can beperformed automatically, as on a Beckman 990 automatic synthesizer,using a program such as that reported in Rivier et al. (1978).

After the desired amino acid sequence has been completed, theintermediate peptide can be removed from the resin support by treatmentwith a reagent, such as liquid hydrogen fluoride or TFA (if using Fmocchemistry), which not only cleaves the peptide from the resin but alsocleaves all remaining side chain protecting groups and also the α-aminoprotecting group at the N-terminus if it was not previously removed toobtain the peptide in the form of the free acid. If Met is present inthe sequence, the Boc protecting group is preferably first removed usingtrifluoroacetic acid (TFA)/ethanedithiol prior to cleaving the peptidefrom the resin with HF to eliminate potential S-alkylation. When usinghydrogen fluoride or TFA for cleaving, one or more scavengers such asanisole, cresol, dimethyl sulfide and methylethyl sulfide are includedin the reaction vessel.

Cyclization of the linear peptide is preferably affected, as opposed tocyclizing the peptide while a part of the peptido-resin, to create bondsbetween Cys residues. To effect such a disulfide cyclizing linkage,fully protected peptide can be cleaved from a hydroxymethylated resin ora chloromethylated resin support by ammonolysis, as is well known in theart, to yield the fully protected amide intermediate, which isthereafter suitably cyclized and deprotected. Alternatively,deprotection, as well as cleavage of the peptide from the above resinsor a benzhydrylamine (BHA) resin or a methylbenzhydrylamine (MBHA), cantake place at 0° C. with hydrofluoric acid (HF) or TFA, followed byoxidation as described above. A suitable method for cyclization is themethod described by Cartier et al. (1996).

Muteins, analogs or active fragments, of the foregoing contulakin-G orThr₁₀-g contulakin-G are also contemplated here. See, e.g., Hammerlandet al (1992). Derivative muteins, analogs or active fragments of theconotoxin peptides may be synthesized according to known techniques,including conservative amino acid substitutions, such as outlined inU.S. Pat. No. 5,545,723 (see particularly col. 2, line 50 to col. 3,line 8); U.S. Pat. No. 5,534,615 (see particularly col. 19, line 45 tocol. 22, line 33); and U.S. Pat. No. 5,364,769 (see particularly col. 4,line 55 to col. 7, line 26), each incorporated herein by reference.

Pharmaceutical compositions containing a compound of the presentinvention as the active ingredient can be prepared according toconventional pharmaceutical compounding techniques. See, for example,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa.). Typically, an antagonistic amount of the activeingredient will be admixed with a pharmaceutically acceptable carrier.The carrier may take a wide variety of forms depending on the form ofpreparation desired for administration, e.g., intravenous, oral orparenteral.

For oral administration, the compounds can be formulated into solid orliquid preparations such as capsules, pills, tablets, lozenges, melts,powders, suspensions or emulsions. In preparing the compositions in oraldosage form, any of the usual pharmaceutical media may be employed, suchas, for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents, suspending agents, and the like in thecase of oral liquid preparations (such as, for example, suspensions,elixirs and solutions); or carriers such as starches, sugars, diluents,granulating agents, lubricants, binders, disintegrating agents and thelike in the case of oral solid preparations (such as, for example,powders, capsules and tablets). Because of their ease in administration,tablets and capsules represent the most advantageous oral dosage unitform, in which case solid pharmaceutical carriers are obviouslyemployed. If desired, tablets may be sugar-coated or enteric-coated bystandard techniques.

For parenteral administration, the compound may be dissolved in apharmaceutical carrier and administered as either a solution or asuspension. Illustrative of suitable carriers are water, saline,dextrose solutions, fructose solutions, ethanol, or oils of animal,vegetative or synthetic origin. The carrier may also contain otheringredients, for example, preservatives, suspending agents, solubilizingagents, buffers and the like. When the compounds are being administeredintrathecally, they may also be dissolved in cerebrospinal fluid.

Administration of the active agent according to this invention may beachieved using any suitable delivery means, including:

(a) pump (see, e.g., Annals of Pharmacotherapy, 27:912 (1993); Cancer,41:1270 (1993); Cancer Research, 44:1698 (1984));

(b), microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888;and 5,084,350);

(c) continuous release polymer implants (see, e.g., U.S. Pat. No.4,883,666);

(d) macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881,4,976,859 and 4,968,733 and published PCT patent applicationsWO92/19195, WO 95/05452);

(e) naked or unencapsulated cell grafts to the CNS (see, e.g., U.S. Pat.Nos. 5,082,670 and 5,618,531);

(f) injection, either subcutaneously, intravenously, intra-arterially,intramuscularly, or to other suitable site; or

(g) oral administration, in capsule, liquid, tablet, pill, or prolongedrelease formulation.

In one embodiment of this invention, an active agent is delivereddirectly into the CNS, preferably to the brain ventricles, brainparenchyma, the intrathecal space or other suitable CNS location, mostpreferably intrathecally.

Alternatively, targeting therapies may be used to deliver the activeagent more specifically to certain types of cells, by the use oftargeting systems such as antibodies or cell-specific ligands. Targetingmay be desirable for a variety of reasons, e.g. if the agent isunacceptably toxic, if it would otherwise require too high a dosage, orif it would not otherwise be able to enter target cells.

The active agents, which are peptides, can also be administered in acell based delivery system in which. a DNA sequence encoding an activeagent is introduced into cells designed for implantation in the body ofthe patient, especially in the spinal cord region. Suitable deliverysystems are described in U.S. Pat. No. 5,550,050 and published PCTApplication Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635.Suitable DNA sequences can be prepared synthetically for each activeagent on the basis of the developed sequences and the known geneticcode.

The active agent is preferably administered in a therapeuticallyeffective amount. The actual amount administered, and the rate andtime-course of administration, will depend on the nature and severity ofthe condition being treated. Prescription of treatment, e.g. decisionson dosage, timing, etc., is within the responsibility of generalpractitioners or specialists, and typically takes into account thedisorder to be treated, the condition of the individual patient, thesite of delivery, the method of administration and other factors knownto practitioners. Examples of techniques and protocols can be found inRemington's Pharmaceutical Sciences. Typically, the active agents of thepresent invention exhibit their effect at a dosage range of from about0.001 μg/kg to about 500 μg/kg, preferably from about 0.01 μg/kg toabout 100 μg/kg, of the active ingredient, more preferably, from about0.10 μg/kg to about 50 μg/kg, and most preferably, from about 1 μg/kg toabout 10 μg/kg. A suitable dose can be administered in multiplesub-doses per day. Typically, a dose or sub-dose may contain from about0.1 μg to about 500 μg of the active ingredient per unit dosage form. Amore preferred dosage will contain from about 0.5 μg to about 100 μg ofactive ingredient per unit dosage form. Dosages are generally initiatedat lower levels and increased until desired effects are achieved.

EXAMPLES

The present invention is further detailed in the following examples,which are offered by way of illustration and are not intended to limitthe invention in any manner. Standard techniques well known in the artor the techniques specifically described below are utilized. Theabbreviations used are: Bop, benzotriazoyloxy-tris (dimethyl amino)phosphonium hexafluorophosphate; Boc, tert butyloxycarbonyl; Fmoc,9-fluoroenylmethoxy carbonyl; Gal, galactose; GalNAc, N-acetylgalactosamine; hNTR1, human neurotensin type 1 receptor; Hex, hexose;HexNAc, N-acetyl hexosamine; icv, intra cerebroventricular; LSI, liquidsecondary ionization; MALD, matrix assisted laser desorption; MS, massspectrometry; mNTR3, mouse neurotensin type 3 receptor; nano-ESI,nano-electrospray; NMP, N-methylpyrrolidone; NMR, nuclear magneticresonance; ppm, parts per million; rNTR1, rat neurotensin type 1receptor; rNTR2, rat neurotensin type 2 receptor; RP-HPLC, reversephase-high performance liquid chromatography. Amino acids are indicatedby the standard three or one letter abbreviations.

Example 1 Experimental Procedures for Initial Analysis of Contulakin-G

1. Crude venom. Conus geographus specimens were collected fromMarinduque Is. in the Philippines. The crude venom was obtained bydissection of the venom duct gland and then freeze dried and stored at−70° C.

2. Peptide purification. Freeze dried C. geographus venom (1 g) wasextracted with 1.1% acetic acid and chromatographed on a Sephadex G-25column eluted with 1.1% acetic acid as previously described (Olivera etal., 1984). A peptide that makes mice sluggish and unresponsive waspurified by a series of RP-HPLC purifications on preparative andsemi-preparative and analytical reverse phase C₁₈ columns. A gradient ofacetonitrile in 0.1% trifluoroacetic acid was used to elute the peptidefrom the columns. The major species was re-purified prior to furthercharacterization. Briefly, one gram of crude lyophilized venom fromConus geographus was extracted and applied on a Sephadex G-25 column aspreviously described (Olivera et al., 1984). Three successive fractionscontaining paralytic and sleeper activities (Ve/Vo=1.37 to 1.41) werepooled, applied on a preparative reversed phase Vydac C₁₈ column andeluted with a gradient of acetonitrile in 0.1% trifluroacetic acid (FIG.1). The component indicated by an arrow in FIG. 1 caused wobbling anddeath when administered icv in mice. This was applied on asemipreparative C₁₈ column, eluted with 12-42% acetonitrile gradient in0.1% trifluroacetic acid. The component which made mice unresponsivewhen administered icv, was further purified with an isocratic elution at20.4% acetonitrile in 0.1% trifluroacetic acid. A mouse injected icvwith an aliquot of the component had trouble righting itself in 5 minand became very sluggish within 12 min. In approximately 25-30 min, themouse was stretched out and laid on its stomach.

3. Bioactivity. Typically, mice injected icv with the partially purifiednative peptide initially had trouble righting after 5 min, becamesluggish after 12 min and then rested on their stomachs after 30 min.These signs were used as an assay to identify the biologically activepeptide during purification.

4. Enzyme hydrolysis. Approximately 180 pmol of the peptide (6 μL) wasincubated with 7 mU β-Galactosidase (bovine testes) (2 μL) in 50 of μL50 mM citrate/phosphate buffer (pH 4.5) for 53 hr at 32° C.Approximately 60 pmol of the peptide (2 μL) was incubated with 2 mUO-glycosidase (Diplococcus pneumoniae) (2 μL) in 50 μL of 20 mMcacodylic acid (pH 6.0) for 19 hr at 32° C.

5. Chemical sequence and amino acid analysis. Automated chemicalsequence analysis was performed on a 477A Protein Sequencer (AppliedBiosystems, Foster City, Calif.). Amino acid analysis was carried outusing pre-column derivatization. Approximately 500 pmol of thecontulakin-G was sealed under vacuum with concentrated HCl, hydrolyzedat 110° C. for 24 hr, lyophilized and then derivatized witho-phthalaldehyde. The derivatized amino acids were then analyzed withRP-HPLC.

6. Mass spectrometry. Matrix assisted laser desorption (MALD)(Hillenkamp et al., 1993) mass spectra were measured using a ‘BrukerREFLEX’ (Bruker Daltonics, Billerica, Ma.) time-of-flight Cotter, 1989)mass spectrometer fitted with a gridless reflectron, an N₂ laser and a100 MHz digitizer. An accelerating voltage of +31 kV and a reflectorvoltage between 1.16 and 30 kV were employed for the post source decay(Spengler et al., 1992) measurements. The sample (in 0.1% aqueoustrifluoroacetic acid) was applied with α-cyano-4-hydroxycinnamic acid.Liquid secondary ionization (LSI) (Barber et al., 1982) mass spectrawere measured using a Jeol HX110 (Jeol, Tokyo, Japan) double focusingmass spectrometer operated at 10 kV accelerating voltage, 1000 or 3000resolution. The sample (in 0.1% aqueous trifluoroacetic acid and 25%acetonitrile) was mixed in a thioglycerol and dithiothreitol matrix.Nano-electrospray (nano-ESI) mass spectra were measured using an Esquireion trap mass spectrometer (Bruker Daltonics, Billerica, Ma.). TheRP-HPLC purified sample, collected in 0.1% aqueous trifluoro-acetic acidand acetonitrile was diluted in methanol 1% acetic acid, transferred toa nanospray capillary and analyzed. The mass accuracy was typicallybetter than 1000 ppm for the time-of-flight instrument, 200 ppm for theion trap instrument and 20-100 ppm for the double focusing massspectrometer depending on the resolving power settings of the magneticsector instrument employed.

7. Synthesis of contulakin-G. The solid-phase glycopeptide synthesis wascarried out manually using Fmoc chemistry, with t-butyl ether side chainprotection for tyrosine and serine, N-t-Boc side chain protection forlysine, and t-butyl ester side chain protection for glutamic acid(protected amino acids were obtained from Bachem, Torrance, Calif.).Starting with a Wang resin, the amino acids were coupled withBop/diisopropylethylamine/N-methylpyrrolidone/dichloro-methane (Stewartet al., 1984; LeNguyen et al., 1986) and the N-deprotections were donewith N-methylpyrrolidone/piperidine (Stewart et al., 1984; LeNguyen etal., 1986). The Wang resin was prepared at The Salk Institute with asubstitution of 0.2 nmol/g. After coupling of the first six amino acids,the resin was coupled with peracetylatedFmoc-Oβ-D-Galp-(1→3)-α-D-GalpNAc-(1→O)-threonine, synthesized asdescribed elsewhere (Luning et al., 1989), followed by single couplingof the remaining nine amino acids in the sequence. Care was taken toremove acetic acid and acetate impurities from the glycosylated aminoacids; this included chromatographic purification on silica gel usingdichloromethane-ethyl acetate 4:1 as eluant, concentration and finallyophilization of the product from benzene. Non-glycosylated peptide wassimilarly synthesized using Fmoc-threonine (Bachem, Torrance, Calif.).The resin was subjected to cleavage conditions (95% trifluoroaceticacid/5% anisole (Stewart et al., 1984)), and in the case of theglycopeptide, the resulting peracetylated glycopeptide was isolated withRP-HPLC, the major component m/z 2322.3 (MALD analysis) corresponding tothe desired product (2322.0 Da). After lyophilization, the peracetylatedglycopeptide was treated with 20 μL of sodium methoxide (Sigma, StLouis, Mo.) (50 mM) in dry methanol for 1 minute (to remove O-acetylgroups on the sugar (Norberg et al., 1994)) and lyophilized at −20° C.The deacetylated sample was loaded onto a Waters Prep LC/System 500Aequipped with gradient controller, Waters Model 450 Variable WavelengthDetector and Waters 1000 PrepPack cartridge chamber column (65.5×320 mm)packed with Vydac C₁₈ 15-20 μm particles. Flow conditions: wavelength230 nm, AUFS 2.0, flow 100 mL/min., gradient 20-60% B/60 min; (where theA buffer was 0.1% trifluoroacetic acid in water and the B buffer was0.1% trifluoroacetic acid in 60% aqueous acetonitrile). The fractions(200 mL) were collected manually. The major component, m/z 2069.9 (LSIanalysis), corresponded to the desired product (2069.98 Da). Afterpreparative RP-HPLC purification, sufficient purified contulakin-G wasobtained for analytical characterization and biological studies. A moreextensive characterization of the synthetic contulakin-G including ¹HNMR data will be presented elsewhere.

8. Co-elution. The native and synthetic contulakin-G were analyzedseparately and co-eluted with RP-HPLC, using a 2.1×150 mm Vydac C₁₈column and a 0.5%/min gradient from 0% B to 40% B (where the A bufferwas 0.55% trifluoroacetic acid in water and the B buffer was 0.05%trifluoroacetic acid in 90% aqueous acetonitrile).

9. Binding studies. The non-glycosylated Thr₁₀-contulakin-G andsynthetic contulakin-G were assayed with the human neurotensin type 1receptor (hNTR1) using a Biomek 1000 robotic workstation for allpipetting steps in the radioligand binding assays, as previouslydescribed (Cusack et al., 1993). Competition binding assays with [³H]neurotensin₁₋₁₃ (1 nM) and varying concentrations of unlabeledneurotensin₁₋₁₃, non-glycosylated Thr₁₀-contulakin-G or syntheticcontulakin-G were carried out with membrane preparations from HEK-293cell line. Nonspecific binding was determined with 1 μM unlabeledneurotensin₁₋₁₃ in assay tubes with a total volume of 1 mL. Incubationwas at 20° C. for 30 min. The assay was routinely terminated by additionof cold 0.9% NaCl (5×1.5 mL), followed by rapid filtration through aGF/B filter strip that had been pretreated with 0.2% polyethylenimine.Details of binding assays have been described before (Cusack et al.,1991). The data were analyzed using the LIGAND program (Munson et al.,1980).

The non-glycosylated Thr₁₀-contulakin-G and synthetic contulakin-G wereseparately assayed with the rat neurotensin type 1 and type 2 receptors(rNTR1 and rNTR2) and mouse neurotensin type 3 receptor (mNTR3).[¹²⁵I-Tyr³] neurotensin₁₋₁₃ was prepared and purified as previouslydescribed (Saadoul et al., 1984). Stable transfected CHO cellsexpressing either the rNTR1 (Tanaka et al., 1990) or the rNTR2 (clonedin the laboratory of J. Mazella by screening a rat brain cDNA library(Stratagene)) were grown in DMEM containing 10% fetal calf serum and0.25 mg/mL G418 (Sigma, France). Cell membrane homogenates were preparedas initially described (Chabry et al., 1994). Protein concentration wasdetermined by the Bio-Rad procedure with ovalbumin as the standard.

10. Binding experiments on cell membranes. Membranes (25 μg for NTR2 and10 μg for NTR1) were incubated with 0.4 nM [¹²⁵I-Tyr³] neurotensin₁₋₁₃(2000 Ci/mmol) and increasing concentrations of Neurotensin₁₋₁₃,non-glycosylated Thr₁₀-contulakin-G or synthetic contulakin-G for 20 minat 25° C. in 250 μl of 50 mM Tris-HCl (pH 7.5) containing 0.1% bovineserum albumin and 0.8 mM 1-10-phenanthroline. Binding experiments wereterminated by the addition of 2 mL of ice-cold buffer followed byfiltration through cellulose acetate filters (Sartorius) and washingtwice. Radioactivity retained on filters was counted with a γ-counter.

11. Binding experiments on solubilized extracts. CHAPS-solubilizedextracts (100 μg) were incubated with 0.2 nM [¹²⁵I-Tyr³] neurotensin₁₋₁₃for 1 hr at 0° C. in 250 μL of Tris-glycerol buffer containing 0.1%CHAPS. Bound ligand was separated from free ligand by filtration on GF/Bfilters pretreated with 0.3% polyethylenimine. Filters were rapidlywashed twice with 3 mL of ice cold buffer and counted for radioactivity.

For binding experiments on mNTR3, membrane homogenates from mouse brainwere re-suspended in 25 mM Tris-HCl buffer (pH 7.5) containing 10% (w/v)glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 1 μM pepstatin, 1 mMiodoacetamide, and 5 mM EDTA (Tris-glycerol buffer). Solubilization wascarried out by incubating homogenates at a concentration of 10 mg/mL inthe Tris-glycerol buffer with 0.625% CHAPS containing 0.125% CHS(Mazella et al., 1988). Solubilized extracts were recovered bycentrifugation at 100,000×g during 30 min at 4° C. and used eitherimmediately or stored at −20° C.

12. Phosphoinositides determination. Cells expressing the rNTR1 or NTR2were grown in 12-well plates for 15-18 hr in the presence of 1 μCi ofmyo-[³H]inositol (ICN) in a serum-free HAM's-F-10 medium. Cells werewashed with Earle buffer, pH 7.5, (25 mM Hepes, 25 mM Tris, 140 mM NaCl,5 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 5 mM glucose) containing 0.1%bovine serum albumin, and incubated for 15 min at 37° C. in 900 μl of 30mM LiCl in Earle buffer. Neurotensin₁₋₁₃ was then added at the indicatedconcentrations for 15 min. The reaction was stopped by 750 μL of icecold 10 mM HCOOH, pH 5.5. After 30 min at 4° C., the supernatant wascollected and neutralized by 2.5 mL of 5 mM NH₄OH. Total[³H]phosphoinositides (PIs) were separated from free [³H]inositol onDowex AG-X8 (Bio-Rad) (Van Renterghem et al., 1988) chromatography byeluting successively with 5 mL of water and 4 mL of 40 mM and 1 Mammonium formate, pH 5.5. The radioactivity contained in the 1 Mfraction was counted after addition of 5 mL of Ecolume (ICN).

13. Identification of a cDNA clone encoding contulakin-G. Contulakin-Gencoding clones were selected from a size-fractionated cDNA libraryconstructed using mRNA obtained from a Conus geographus venom duct aspreviously described (Colledge et al., 1992). The library was screenedusing a specific probe corresponding to amino acids #10-15 of thepeptide (5′-ATR ATN GGY TTY TTN GT-3′; SEQ ID NO:3). The oligonucleotidewas end-labeled, hybridized and a secondary screening by polymerasechain reaction was performed on 10 clones that hybridized to this probeas previously described (Jimenez et al., 1996). Clones identified in thesecondary screen were prepared for DNA sequencing as previouslydescribed (Monje et al., 1993). The nucleic acid sequence was determinedaccording to the standard protocol for Sequenase version 2.0 DNAsequencing kit as previously described (Jimenez et al., 1996).

Example 2 Purification of Contulakin-G

A fraction of Conus geographus venom was detected which made miceexceedingly sluggish. Normally, when mice that are sitting down arepoked with a rod, they immediately get up and run a considerabledistance. Upon i.c.v. injection of the fraction from Conus geographusindicated in FIG. 5, the mice had to be poked with much more forcebefore they got up at all, and after getting up, they would walk one ortwo steps and immediately sit down again. This “sluggish behavior” wasfollowed through several steps of purification, and the apparentlyhomogeneous peptide was further analyzed. This peptide was designatedcontulakin-G (the Filipino word tulakin{acute over ( )} means “has to bepushed or prodded,” from the root word tulak, to push). The “G”indicates that the peptide is from Conus geographus.

Example 3 Biochemical Characterization of the Purified Contulakin-G

Attempted amino acid sequence analysis of the purified peptide revealedthat the peptide was blocked at the N-terminus. Since most N-terminallyblocked Conus peptides have a pyroglutamate residue at position 1, thepeptide was treated with pyroglutamate aminopeptidase. This resulted ina shift in retention time suggesting removal of a pyroglutamate residue.After enzyme treatment, the sequenceSer-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Xaa-Lys-Lys-Pro-Tyr-Ile-Leu (SEQ IDNO:4) was obtained by standard Edman methods confirming removal of thepyroglutamate residue, where Xaa indicates no amino acid was assigned inthe 9th cycle (at position 10) although a very low signal for threoninewas obtained. Amino acid analyses were consistent with the presence ofone threonine residue in the peptide.

In order to confirm the nature of the amino acid residue in position 10,a cDNA clone encoding the peptide was isolated. The nucleotide sequenceand presumed amino acid sequence revealed by the clone are shown inTable 1 and in SEQ ID NO:5 and SEQ ID NO:6, respectively. The amino acidsequence of contulakin-G obtained by direct Edman sequencing is foundencoded towards the C-terminal end of the only significant open readingframe in the clone (at residues 51-66); the predicted amino acidsequence reveals that position 10 of the mature peptide (residue 60 ofthe precursor) is encoded by a codon for threonine. Thus, the Edmansequencing, together with cloning results, suggested that a modifiedthreonine residue was present in position 10.

TABLE 1 DNA (SEQ ID NO:5) and Peptide (SEQ ID NO:6) Sequences ofContulakin-G atg cag acg gcc tac tgg gtg atg gtg atg atg atg gtg tgg attgca Met Gln Thr Ala Tyr Trp Val Met Val Met Met Met Val Trp Ile Ala gcccct ctg tct gaa ggt ggt aaa ctg aac gat gta att cgg ggt ttg Ala Pro LeuSer Glu Gly Gly Lys Leu Asn Asp Val Ile Arg Gly Leu gtg cca gac gac ataacc cca cag ctc atg ttg gga agt ctg att tcc Val Pro Asp Asp Ile Thr ProGln Leu Met Leu Gly Ser Leu Ile Ser cgt cgt caa tcg gaa gag ggt ggt tcaaat gca acc aag aaa ccc tat Arg Arg Gln Ser Glu Glu Gly Gly Ser Asn AlaThr Lys Lys Pro Tyr att cta agg gcc agc gac cag gtt gca tct ggg cca tagIle Leu Arg Ala Ser Asp Gln Val Ala Ser Gly Pro

Mass spectrometric analyses (MALD, LSI and nano-ESI) of the purifiedcontulakin-G fraction revealed a variety of intact species as summarizedin Table 2. Some variation in the intensity of the different species wasobserved with different ionization techniques, which was ascribed todifferences in the bias (Craig et al., 1994) with each ionizationtechnique. In the following analysis, we have concentrated on the majorglycoform with intact mass M₁=2069 observed with all of the ionizationtechniques investigated. The difference between the observed mass (2069Da) and the mass calculated for the sequence assuming Thr at residue 10(1703.83 Da) was 365 Da. Because one possible modification of threonineis O-glycosylation, we proposed, based on this mass difference, that theunidentified residue was hexose-N-acetyl-hexosamine-threonine(Hex-HexNAc-Thr) which would result in the addition of 365.13 Da. Theobserved masses (Table 2) are consistent with the calculatedmonoisotopic mass of the [M₁+H]⁺ or [M₁+2H]²⁺ of the proposeddisaccharide-linked peptide (2069.98 or 1035.5 Da respectively). Intensefragment ions were observed in the nano-ESI MS/MS mass spectrum of thedoubly charged [M₁+2H]²⁺ intact molecule ion of contulakin-G (FIG. 6)corresponding to the loss of the complete Hex-HexNAc glycan (denotedp(χ₃)₁₀ (Craig et al., 1993) or loss of the terminal hexose residue(p(χ₈)₁₀).

TABLE 2 Species Observed with MALD, LSI and nano-ESI Analysis ofPurified Contulakin-G Molecule Species M₁ (Da) M₂ (Da) M₃ (Da) M₄ (Da)Proposed glycan HexHexNAc SO₄HexHexNAc Hex₃ Hex₂HexNAc₂ MALD/TOF2069^(a) — 2186^(b) — LSI/Magnetic 2068.7 2149.6 — 2436.5 nano-ESI/IT2068.6^(c) 2148.6^(c) — — Mono^(d) [M + H]⁺ 2068.97 2148.92 2189.942434.10 Average^(e) [M + H]⁺ 2070.19 2150.25 2191.27 2435.53 ^(a)m/z2091 and 2107 corresponding with [M₁ + Na]⁺ and [M₁ + K]⁺ were alsoobserved. ^(b)m/z 2210 and 2225 corresponding with [M₃ + Na]⁺ and [M₃ +K]⁺ were also observed. ^(c)m/z 1035.3 and 1075.3 doubly charged ionswere observed. ^(d)monoisotopic [M + H]⁺ masses were calculated based onproposed glycan and contulakin-G sequence. ^(e)average [M + H]⁺ masseswere calculated based on proposed glycan and contulakin-G sequence.

Example 4 Evidence that Thr-10 is O-glycosylated

Native contulakin-G was treated with β-galactosidase isolated frombovine testes. This enzyme preferentially hydrolyzes terminal β1→3galactopyranosyl residues from the non-reducing end of glycoconjugates.After β-galactosidase treatment of the native sample a new component wasobserved on RP-HPLC. This component was collected and analyzed withMALD-MS in which a species was observed at m/z 1907. The difference inmass and the specificity of the enzyme are consistent with a terminalgalactose residue being released. Based on the β-galactosidasehydrolysis results we reasoned that the glycan moiety might besusceptible to O-glycosidase treatment, which liberates the disaccharideGal (β1→3) GalNAc (α1→) bound to serine or threonine as a core unit ofglycopeptides. O-glycosidase treatment of the native contulakin-G did infact result in a new species after the enzyme hydrolysis mixture wasanalyzed on RP-HPLC. The new component was collected and analyzed withMALD-MS where an m/z 1704 species was observed consistent with loss ofHex-HexNAc (i.e., the mass was consistent with that predicted for thepeptide with an unmodified threonine residue at position 10). The enzymehydrolysis results are consistent with the presence of a Gal (β1→3)GalNAc (α1→) glycan. Based on the O-glycosidase and the β-galactosidasehydrolysis results, the structure of the most abundant glycopeptide is:

              Gal (β1 − >3) GalNAc (α1 − >)                                     |pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH (SEQID NO:_)

Example 5 Synthesis of the Non-Glycosylated and GlycosylatedContulakin-G

The 16-amino acid non-glycosylated peptide was chemically synthesized.The synthetic material was found to have the same retention time as theenzymatically des-glycosylated contulakin-G on RP-HPLC. The 16-aminoacid glycosylated contulakin-G containing Gal (β1→3) GalNAc (α1→)attached to Thr₁₀ was also synthesized. This synthetic glycosylatedcontulakin-G co-eluted with the native contulakin-G on RP-HPLC. The postsource decay fragmentation spectra observed for both native andsynthetic contulakin-G showed very similar fragmentation patterns.

Example 6 Biological Potency of Synthetic Glycosylated andNon-Glycosylated Contulakin-G

The loss of motor control for which the native contulakin-G wasoriginally isolated, together with gut contraction, absence ofpreening/grooming, and reduced sensitivity to tail depression were signsobserved when Neurotensin₁₋₁₃, non-glycosylated Thr₁₀-contulakin-G orsynthetic contulakin-G were administered icv. In order to investigatethese observations in more detail, a dose response comparison wasperformed as detailed in Table 3. While the non-glycosylatedThr₁₀-contulakin-G analog was active at doses of 1 nmol and higher, itwas inactive at 300 pmol doses. In contrast, contulakin-G was found toelicit loss of motor control at doses of 30 pmol or approximately 5pmol/g.

TABLE 3 Effect of icv administration of Neurotensin₁₋₁₃,Thr₁₀-Contulakin-G and Contulakin-G in 14-18 day old mice Av. Time ofsymptom dose Number age weight A^(c) B^(c) R^(c) Compound (pmol) ofmice^(a) (days) (g)^(b) (min) (min) (min) NSS   0 8 16 6.2 —^(d) —^(d)—^(d) neurotensin₁₋₁₃ 1000 2 14 7.1 9 23 >120 Thr₁₀-contulakin-G 1000 716.6 6.5 9 99 159 Thr₁₀-contulakin-G  300 6 15.7 6.1 —^(d) —^(d) —^(d)contulakin-G 1000 6 18 7.1 1.0 120 187 contulakin-G  300 8 15.5 6.6 2.942 151 contulakin-G  100 8 15.9 6.6 2.8 40 136 contulakin-G  30 7/9 156.3 5.3 23 114 ^(a)number of mice affected. ^(b)average age (days) andweight (g). ^(c)average time after which specific behavior observed(observations were made every 2 min for first 15 min and every 15 minthereafter). Symptoms A and B were observed when a mouse was placed ontoa bench top after lifting it by the tail for a second. Symptom A: Themouse moved at most a few steps and rested with the hind legs spreadout. Mouse remained stationary unless pushed or lifted. Symptom B: Themouse remained sluggish but the position of the hind part of the bodywhen at # rest resembled that of the NSS controls. Symptom R: Recovery,the mouse moved freely when released. ^(d)no symptom observed.

The six C-terminal amino acids of contulakin-G show significantsimilarity to the sequences of neurotensin₁₋₃, neuromedin, xenin and theC-terminus of xenopsin (see Table 4). Because of the similar signsobserved when either contulakin-G or Neurotensin₁₋₃ were administeredicv and the significant homology between contulakin-G and Neurotensin₁₋₃we tested the affinity of contulakin-G for a number of the clonedneurotensin receptors. As shown in Table 5, the non-glycosylatedThr₁₀-contulakin-G analog was found to bind the human neurotensin type Ireceptor (hNTR1) with 10 fold lower affinity than Neurotensin_(3,) andeven lower affinities for the other NTR's. Contulakin-G exhibitedsignificantly lower affinity than the non-glycosylatedThr₁₀-contulakin-G analog for all of the NTR's tested.

Both contulakin-G and the non-glycosylated Thr₁₀-contulakin-G analogacted as agonists when tested on CHO cells expressing the rNTR1. Noresponse was observed with CHO cells expressing the rNTR2. Thenon-glycosylated Thr₁₀-contulakin-G analog resulted in slightly lowerpotency (0.6 nM) but with similar efficacy as compared withNeurotensin₁₋₁₃. The synthetic glycosylated contulakin-G potency wassignificantly lower (20-30 nM) and the agonistic efficacy wasapproximately half that observed for Neurotensin₁₋₁₃.

TABLE 4 Sequence Comparison of Contulakin-G and Members of theNeurotensin Family of Peptides Name Sequence (SEQ ID NO:) Id^(a) Si^(b)Source Ref Contulakin- <ESEEGGSNAT*KKPYIL-OH — — C. geographus G (7)neurotensin <ELYENKPRRPYIL-OH 66 33 bovine (1) (8) hypothalamusneuromedin KIPYIL-OH 83 0 porcine spinal cord (2) N (9) xenopsinQGKRPWIL-OH 66 16 Xenopus laevis (3) (10) xeninMLTKFETKSARVKGLSFHPKRPWIL-OH 66 16 human gastric (4) (12) mucosa*indicates an O-linked glycosylated threonine/serine residue.^(a)percentage identity of the 6 C-terminal amino acids compared tocontulakin-G₁₁₋₁₆. ^(b)percentage similarity of the 6 C-terminal aminoacids compared to contulakin-G₁₁₋₁₆. References (1) Carraway et al.,1973; (2) Minamino et al., 1984; (3) Araki et al., 1973; (4) Feurle etal., 1992.

TABLE 5 Comparison of Binding Affinity of Neurotensin₁₋₁₃,Thr₁₀-Contulakin-G and Contulakin-G for the Cloned Human and RatNeurotensin Type 1 Receptor (NTR1), the Rat Neurotensin Type 2 Receptor(rNTR2), and the Solubilized Mouse Neurotensin Type 3 Receptor (mNTR3)IC₅₀ (nM) Receptor Compound hNTR1 rNTR1 rNTR2 mNTR3 neurotensin₁₋₁₃ 1.43.2 6.0 1.4 Thr₁₀-contulakin-G 23 79 170 71 contulakin-G 960 524 730 250

Example 7 Biological Activity of Contulakin-G Analogs

The biological activity of several peptide analogs of contulakin-G wastested in a similar manner as described above by icv injection in mice.These peptides were synthesized as described herein, and include thefollowing analogs:

Ser₁₀-contulakin-G containing the native glycosylation on Ser₁₀ (analogA); and

Δ1-9-Ser₁₀-contulakin-G containing the native glycosylation on Ser₁₀(analog B) It was found that analog A was slightly more active than thenative contulakin-G. It was also found that analog B had the sameactivity, i.e., onset and recovery time than analog A when tested in twoweek old mice at a dose of 100 pmole. In this test, the mice were stillnot able to right themselves after 75 minutes. When tested in three weekold mice at doses of 1 nmole and 300 pmole, the same activity was seenbetween the analogs and these mice were drowsy for 100 minutes. Theseexperiments demonstrate that glycosylated contulakin-G analogs in whichN-terminal amino acids residues have been removed, retain activity.Similar results are achieved for other analogs, such asΔ1-5-Ser₆-contulakin-G containing the native glycosylation on Ser₆ withor without the native glycosylation on Thr₁₀. These results show thatthe placement of a glycosylated serine residue proximal to the site oftruncation yields active analogs.

The Conus peptide characterized above, contulakin-G, has a novelbiochemical feature: a post-translationally O-glycosylated threonine notpreviously found in Conus peptides. Using mass spectrometry and specificenzymatic hydrolyses, it was found that Thr₁₀ was modified with thedisaccharide Gal (β1→3) GalNAc (α1→). The corresponding glycosylated andnon-glycosylated forms of contulakin-G were synthesized which confirmedthe molecular structure of this major glycosylated form of the nativemolecule based on RP-HPLC co-elution and MS fragmentation criteria. Themasses of the other more minor molecule species observed with massspectrometry are consistent with glycan structural variations atperipheral sites on the characterized oligosaccharide core unit(Baenziger, 1994).

An analysis of a cDNA clone encoding contulakin-G reveals that theprepropeptide organization of the contulakin-G precursor is similar tothat of other Conus peptide precursors (Olivera et al., 1997). A typicalsignal sequence is found, and immediately N-terminal to the contulakin-Gsequence are two basic amino acids which presumably signal a proteolyticcleavage to generate the N-terminus of the mature peptide (the glutamineresidue would cyclize to pyroglutamate either spontaneously or due tothe action of glutaminyl cyclase (Fischer et al., 1987)). Although inmost respects the contulakin-G precursor has the same organization asall other Conus venom peptide precursors and would be predicted to beprocessed in the same way, the ten C-terminal amino acids predicted bythe clone are not present in contulakin-G purified from venom. Onepossibility is that the clone represents a different variant, forexample one which was alternatively spliced. Alternatively, furtherproteolytic processing at the C-terminus may be required to generatemature contulakin-G.

Over the last 20 years an increasing number of biologically importantglycopeptides and glycoproteins have been identified. Vespulakinin 1,first identified by Pisano et al. (Yoshida et al., 1976), is, to ourknowledge, the only other O-glycosylated peptide toxin which has beenisolated from venom other than Conus. Vespulakinin 1 was extracted fromthe venom sacs of the yellow jacket wasp, Vespula maculifrons. Thepeptide (TAT*T*RRRGRPPGFSPFR-OH (SEQ ID NO:12) where the asteriskindicates an O-linked glycosylated threonine residue) contains twosequential sites of O-linked glycosylation. The C-terminus ofVespulakinin is identical to the sequence of Bradykinin (RPPGFSPFR-OH(SEQ ID NO:13)) and the peptide was found to elicit a number of signsalso elicited by Bradykinin. Vespulakinin is therefore another exampleof an O-linked glycosylated peptide toxin in which the C-terminusappears to target a mammalian neurotransmitter receptor. Thus, bothcontulakin-G and Vespulakinin 1 contain glycosylated N-terminalextensions to sequences with very high homology to mammalianneuropeptides. κA-conotoxin SIVA, a K⁺ channel inhibitor is unusualamong disulfide-rich Conus peptides in having a long N-terminal tail,which has an O-glycosylated residue (Craig et al., 1998).

For most Conus peptides, a specific conformation appears to bestabilized either by multiple disulfide linkages or by the appropriatespacing of γ-carboxyglutamate residues to promote formation of α-helices(Olivera et al., 1990). Conus peptides without multiple disulfidescomprise a most eclectic set of families, including the conopressins,conantokins, contryphans and now contulakin-G. The conopressins areprobably endogenous molluscan peptides, clearly homologous to thevassopressin/oxytocin family of peptides; these are more widelydistributed in molluscan tissues than in Conus venom ducts. However, theother non-disulfide-rich peptides (conantokins, contryphans andcontulakin-G) may be specialized venom peptides exhibiting unusualpost-translational modifications. In addition to the O-glycosylatedthreonine moiety of contulakin-G described here, γ-carboxylation ofglutamate residues and the post-translational epimerization andbromination of tryptophan residues were discovered in conantokins andcontryphans.

Several lines of evidence are consistent with contulakin-G being thefirst member of the neurotensin family of peptides to be isolated froman invertebrate source. First, the C-terminal region of contulakin-Gexhibits a striking degree of similarity to other members of theneurotensin family (all from vertebrates), as shown in Table 4.Furthermore, it was shown above that contulakin-G competes for bindingto three known neurotensin receptor subtypes; evidence that contulakin-Gacts as an agonist on a cloned neurotensin receptor is also presentedabove. Most convincingly however, when contulakin-G is injected intomice, the same behavioral signs are elicited with administration ofneurotensin. Thus, structural data, binding data and in vivo behavioralsymptomatology are all consistent with the assignment of contulakin-G tothe neurotensin family of peptides.

Clearly, both contulakin-G and the non-glycosylated Thr₁₀-contulakin-Gare rNTR1 agonists at physiologically relevant concentrations (20-30 and0.6 nM, respectively). The observed agonistic effects of bothcontulakin-G and the non-glycosylated analog, as well as the absence ofany agonistic effect of these ligands on CHO cells expressing rNTR2using the IP accumulation assay does not correlate with the in vitrobinding data; both peptides are agonists at concentrations significantlybelow their IC₅₀ binding affinity (524 and 79 nM, respectively). Mostunexpected therefore, given its apparently lower binding affinity, isthe increased potency of glycosylated contulakin-G compared with thenon-glycosylated analog after icv administration.

Thus, the role of the glycan is somewhat paradoxical. In vitro, theglycan neither increases the binding affinity, the agonistic potency noragonistic efficacy. In contrast, in vivo, the glycan significantlyincreases the potency of the peptide. One simple explanation is that theincreased potency of contulakin-G compared with the non-glycosylatedanalog is due to increased stability. An alternative mechanism for theincreased potency is transport to the site of action facilitated by theglycan. Additionally, the glycosylated peptide may act with highaffinity on an as-yet-undefined neurotensin receptor subtype (Tyler etal., 1998), or may be a selective high affinity ligand for a particularstate of a neurotensin receptor subtype. Yet another possibility is thatthe relevant targeted neurotensin receptors may be closely co-localizedwith carbohydrate binding sites, and that the glycan may serve as an“address label”, a mechanism postulated for certain opiate peptides.Preliminary data supporting the increased stability hypothesis has beenobtained—proteolytic degradation of contulakin-G is inhibited by thepresence of the glycan moiety. The increased stability may well resultin an enhanced supply of the glycopeptide at the receptor. However, theincreased in vivo potency of contulakin-G conferred by O-glycosylationclearly requires a more balanced evaluation of the possibilitiesoutlined above.

Example 8 Materials and Methods for Assessing Analgesic Activity ofThr₁₀-Contulakin-G

1. Acute pain (hotplate). Thr₁₀-contulakin-G (CGX-1063) or vehicle wasadministered via intracerebroventricular (icv) in a volume of 5 μl.Fifteen minutes after injection, animals were placed on a 55° C.hotplate. The latency to the first response (flinch), a spinallymediated behavioral response, and the first hindlimb lick, a centrallyorganized motor response to acute pain, were recorded. Mice were removedfrom the hotplate after 60 seconds if no response was observed.Immediately prior to being placed on the hotplate, motor function wastested by determining the latency to first fall from an acceleratingrotarod.

2. Persistent pain (formalin test). Intrathecal (it) drug injectionswere performed as described by Hyldon and Wilcox (1980). CGX-1063 (10 or100 pmol) or vehicle was administered in a volume of 5 μl. Fifteenminutes after the it injection, the right hindpaw was injected with 20μl of 5% formalin. Animals were placed in clear plexiglass cylindersbacked by mirrors to facilitate observation. Animals were closelyobserved for 2 minutes per 5 minute period, and the amount of time theanimal spent licking the injected paw was recorded in this manner for atotal of 45-50 minutes. Results are expressed as licking time in secondsper five minutes. At the end of the experiment, all animals were placedon an accelerating rotorod and the latency to first fall was recorded.

2. Neuropathic pain. The partial sciatic nerve ligation model was usedto assess the efficacy of CGX-1063 in neuropathic pain. Nerve injury wasproduced according to the methods of Malmberg and Basbaum (1998).Animals were anesthetized with a ketamine/xylazine solution, the sciaticnerve was exposed and tightly ligated with 8-0 silk suture around ⅓ to ½of the nerve. In sham-operated mice the nerve was exposed, but notligated. Animals were allowed to recover for at least 1 week beforetesting was performed. On the testing day, mice were placed inplexiglass cylinders on a wire mesh frame and allowed to habituate forat least 60 minutes. Mechanical allodynia was assessed with calibratedvon Frey filaments using the up-down method as described by Chaplan etal. (1994), and the 50% withdrawal threshold was calculated. Animalsthat did not respond to any of the filaments in the series were assigneda maximal value of 3.6 grams, which is the filament that typicallylifted the hindlimb without bending, and corresponds to approximately{fraction (1/10)} the animal's body weight.

Example 9 Analgesic Activity of Thr₁₀-Contulakin-G

CGX-1063 (10 fmol-10 mnol, icv) dose-dependently increased the latencyto the first hindpaw lick and first response elicited by the hotplate(FIGS. 7A-7B). Of interest is the difference in potency of CGX-1063 inincreasing the latency to the first hindpaw lick compared to the latencyto first response. CGX-1063 also dose-dependently decreased the latencyto first fall on the rotarod (FIG. 7C). However, this apparent motorimpairment did not appear to be the result of the loss of motorfunction, since animals were capable of normal locomotor activity whenstimulated. Thus, the effect of CGX-1063 on the hotplate unequivocallywas an analgesic effect.

CGX-1063 (10 or 100 pmol, it) dose-dependently and significantlydecreased the second phase of the formalin test (FIG. 8A).Interestingly, the lower dose (10 pmol) was more effective in decreasingthe first phase response time than was the higher dose. This will beexamined in more detail in future experiments. After it administration,CGX-1063 treated animals showed no motor impairment compared to vehicletreated animals (FIG. 8B), indicating that the effect of icv CGX-1063(observed in the hotplate test above) on motor impairment is mediated athigher brain regions, not spinally, and that the analgesic effects ofCGX-1063 can be separated from the motor toxicity by using this route(it) of administration. The downward shift in the rotorod scorescompared to those from animals used in the hotplate test reflects anoverall impairment in these animals due to formalin-induced allodyniaand inflammation of the hindpaw.

One week after partial sciatic nerve ligation, animals showed a markeddecrease in the paw withdrawal threshold on the operated side(ipsilateral) relative to the unoperated side (contralateral),indicating an increase in sensitivity to mechanical stimuli (FIG. 9).Intrathecal administration of CGX-1063 (100 pmol) dramatically increasedthe withdrawal threshold on the ligated side (an approximate six foldincrease). Interestingly, the mechanical threshold on the contralateralside was not significantly altered. In sham-operated animals, there wasno difference in withdrawal threshold between operated and un-operatedsides. After intrathecal CGX-1063, the withdrawal threshold wasuniformly increased in both hindpaws of these animals.

The present data demonstrate that CGX-1063 has potent analgesicproperties in three commonly used models of pain: acute,persistent/inflammatory and neuropathic pain models. CGX-1063administered centrally (icv) dose-dependently reduced the responselatency in the hot plate model of acute pain, and was effective in thelow picomole to high femtomole range. Preliminary data indicate that theanalgesic effect of CGX-1063 in this model is not mediated through anopioid mechanism. CGX-1063 was also effective in reducing nociceptiveactivity in the formalin model of persistent/inflammatory pain. CGX-1063dose-dependently reduced the second (inflammatory) phase of the formalintest, while at the lower dose, reduced phase one activity. Finally,CGX-1063 showed profound analgesic activity in a model of neuropathicpain. Mechanical withdrawal thresholds in this model were increasednearly six fold compared to pre-treatment values, while not alteringsensitivity in the non-injured paw, possibly indicating that CGX-1063reduces neuropathic allodynia while not affecting normal sensorytransmission.

Example 10 Materials and Methods for Assessing Analgesic Activity ofContulakin-G

1. Acute pain (tail-flick). Drug (contulakin-G (CGX-1160) orThr₁₀-contulakin-G (CGX-1063)) or saline was administered intrathecally(i.t.) according to the method of Hylden and Wilcox (Hylden and Wilcox,1980) in a constant volume of 5 μl. Mice were gently wrapped in a towelwith the tail exposed. At various time-points following the i.t.injection, the tail was dipped in a water bath maintained at 54° C. andthe time to a vigorous tail withdrawal was recorded. If there was nowithdrawal by 8 seconds, the tail was removed to avoid tissue damage.

2. Persistent pain (formalin test). CGX-1160, CGX-1063 (1, 10 or 100pmol), neurotensin (NT) (1, 10, 100 or 10000 pmol), or vehicle wasadministered i.t. in a volume of 5 μl. Fifteen minutes after the i.t.injection, the right hindpaw was injected with 20 μl of 5% formalin.Animals were placed in clear plexiglass cylinders backed by mirrors tofacilitate observation. Animals were closely observed for two minutesper five minute period, and the amount of time the animal spent lickingthe injected paw was recorded in this manner for a total of 45-50minutes. Results are expressed as licking time in seconds per fiveminutes. At the end of the experiment, all animals were placed on anaccelerating rotorod and the latency to first fall was recorded.

3. Chronic inflammatory allodynia (CFA model). Mice were givenintraplantar (i.pl.) injections of 20 μl of CFA into the right hindpawand returned to their home cage. Three days later mice were placed inplexiglass cylinders on a wire mesh frame and allowed to habituate forat least 60 minutes. Mechanical allodynia was assessed with calibratedvon Frey filaments using the up-down method as described (Chaplan etal., 1994), and the 50% withdrawal threshold was calculated. Animalsthat did not respond to any of the filaments in the series were assigneda maximal value of 3.6 grams, which is the filament that typicallylifted the hindlimb without bending, and corresponds to approximately{fraction (1/10)} of the body weight.

4. Toxicity testing. To accurately assess the motor impairing effects ofCGX-1160, CGX-1063, and NT, 50 mice were divided into groups receivingi.t. CGX-1160 or CGX-1063 (1, 10, 100, 500 and 1000 pmol), NT (0.1, 1,10, and 100 nmol), or saline (n=5 per group except for the highest doseof each compound where n=3). Starting at 15 minutes post injectionanimals were place on an accelerating rotorod and the latency to firstfall was recorded. Animals were retested at 30, 60, 120, 240 and 300minutes (or until the latency to fall had returned to control values).Rectal temperature was also recorded in these animals at the same timepoints.

Example 11 Analgesic Activity of Contulakin-G

CGX-1160 dose-dependently increased the tail-flick latency (FIG. 10A)with a time to peak effect of ≦30 minutes (the earliest time tested,FIG. 10B). Furthermore, the increase in latency was long-lasting withelevated withdrawal times at 5 hour post injection that returned tobaseline at 24 hours post injection (FIG. 10B). CGX-1063 also showed adose-dependent, though more variable increase in withdrawal latency, andshowed only modest antinociceptive efficacy in this model relative toCGX-1160 (FIGS. 10A-10B). In comparison, NT did not significantlyelevate withdrawal latency in the tail-flick assay (FIGS. 10A-10B).

All of the compounds tested dose-dependently showed antinociceptiveproperties in both phases of the formalin test, but with differentpotencies. CGX-1160 was the most potent of the three compounds. In phase1 of the formalin test (FIG. 11A), CGX-1160 had an ED₅₀ of approximately30-40 pmol while NT had an ED₅₀ of ≈1 nmol. CGX-1063 did not reach the50% antinociception threshold in phase 1, however, the irregulardose-response in this test warrants repeating the 100 pmol dose in thisassay. In phase 2 of the formalin test, all three compoundsdose-dependently reduced the paw licking time (indicated in the figuresas an increase in the percent antinociception; FIG. 11B). Again,CGX-1160 was more potent than the other compounds with an estimated ED₅₀of 1 pmol. Lower doses of this compound will be assessed in the futureto complete the dose response curve necessary to calculate a moreprecise ED₅₀. CGX-1063 was also effective in reducing nociceptivebehavior in phase 2, with an estimated ED₅₀ of 10-20 pmol. NT wasdramatically less potent than either of the contulakins with anestimated ED₅₀ of 600-700 pmol (FIG. 11B).

CGX-1160 showed extremely potent and dose-dependent reversal ofCFA-induced mechanical allodynia (FIG. 12A). One-hundred (100) fmol ofCGX-1160 given i.t. completely reversed the CFA-induced mechanicalallodynia. Interestingly, at this dose, the contralateral sensitivity tomechanical pressure was unaltered indicating a potential unilateralalteration in NT receptors in chronic inflammation. At higher doses ofCGX-1160, the mechanical withdrawal threshold in both the CFA-injectedpaw and the contralateral uninjected paw was dramatically elevated. InFIGS. 12A and 12B, the numbers over the bars indicate the percentincrease in mechanical threshold relative to the pre-drug level. Asindicated, at all doses tested, CGX-1160 had a much greaterantiallodynic effect on the CFA injected side relative to the uninjectedside. CGX-1063 was less potent than CGX-1160, but also completelyreversed the CFA-induced allodynia (FIG. 12B). The minimally effectivedose was 10 pmol, however, at this dose, unlike CGX-1160, thecontralateral side was also elevated relative to pre-drug baselinemeasurements. Consistent with the other models examined in this study,NT showed efficacy in the CFA model at 1 nmol, but not at 100 pmol (FIG.12C). Other doses of CGX-1160 and NT will be examined in the future todetermine accurate ED₅₀s for these compounds.

CGX-1160, -1063, and NT all showed dose-dependent effects on locomotorimpairment and body temperature. For all three compounds, maximalimpairment was at 15 minutes post i.t. injection (locomotor impairment,FIG. 13A) or 30 minutes (hypothermic effects, FIG. 14A). CGX-1063 had nomotor toxicity at the lowest dose tested (1 pmol, FIG. 13B), but athigher doses animals showed significant motor toxicity (estimated TD₅₀of 10 pmol, FIGS. 13B and 15A). At 10 pmol this toxicity lasted for 30minutes, but resolved by 60 minutes. When 100 pmol or 1 nmol wasadministered, animals were motor impaired for 2-3 hours (FIG. 14A).CGX-1160 was equipotent to CGX-1063 in causing motor impairment(estimated TD₅₀ of 10-20 pmol, FIG. 13B). Similar to CGX-1063, at higherdoses (100-500× its ED₅₀) CGX-1160 showed motor impairment that resolvedafter 5 hours (FIGS. 13A and 14B). The estimated TD₅₀ for NT-inducedmotor impairment was 3 nmol (FIG. 13B). Similar to the contulakins, athigh doses, NT-induced motor impairment that lasted 2-4 hours (FIGS. 13Aand 14C).

The hypothermic effects of these compounds were similar to motortoxicity. All three caused a dose-dependent decrease in bodytemperature. CGX-1160 and -1063 were equipotent with an estimated TD₅₀of 100 pmol (FIG. 15B). However, at this dose CGX-1063 induced a drop inbody temperature lasting 2-3 hours (FIGS. 15A and 16A), while thehypothermic effect caused by CGX-1160 resolved by 60 minutes (FIGS. 15Aand 16B). At the highest dose of CGX-1160 (500 pmol, 500× the ED₅₀), thehypothermic effect had not resolved by six hours post-injection (FIG.16B). NT showed a very similar dose-response and time course to thecontulakins. At the lower doses, NT had no effect or showed a shortlasting hypothermic effect (FIG. 16C). At the highest dose, however (100nmol), NT caused a dramatic and long-lasting hypothermia that had notresolved by three hours (FIGS. 15A and 16C).

The present data show that CGX-1160 and CGX-1063 are potent,broad-spectrum analgesic agents effective in several animal models ofacute and chronic pain. CGX-1160 is typically 10 fold more potent thanCGX-1063, and 1000 times more potent than NT (Table 6). CGX-1160 isparticularly potent in the model of chronic inflammatory pain whereCGX-1160 selectively increases the mechanical withdrawal threshold onlyin the paw receiving the CFA injection, while not altering the thresholdof the uninjected paw. This finding indicates that chronic inflammationmay lead to a reorganization of NT receptors in nociceptive pathwayscorresponding to the inflamed paw. Since CGX-1160 was the only compoundin these experiments to show an increased potency, this may indicate anupregulation of a receptor subtype for which CGX-1160 may haveparticular selectivity and specificity. In support of this hypothesis ofCGX-1160 subtype selectivity are the findings that this compound showsantinociception at doses 10-100 fold less than for either locomotorimpairment or hypothermia, whereas CGX-1063 and NT causeantinociception, locomotor impairment, and hypothermia at approximatelyequal doses when administered i.t. Particularly interesting is thelong-lasting hypothermic effect of CGX-1063. When given i.t. at 100 pmol(approximately 10 times its ED₅₀ in phase 2 of the formalin test, seeFIG. 16A), CGX-1063 caused long-lasting hypothermia relative tocomparable antinociceptive doses of CGX-1160 (compare the 10 pmol dosein FIG. 16B) and NT (compare the 10 nmol dose in FIG. 16C). Thispotentially indicates that CGX-1063 is selective for the NT receptorsubtype involved in the hypothermic effect of NT analogs. Thus theO-glycosylation of Thr₁₀ in CGX-1160 may impart selectivity for theantinociceptive NTR subtype, currently thought to be NTR2, as well asmetabolic resistance to peptidases.

TABLE 6 Comparison of the Antinociceptive Effects, Motor ImpairmentEffects, and Protective Index of CGX-1160, CGX-1063, and NT in theFormalin Test (phase 2) and CFA-Induced Allodynia Test Compound ED₅₀,pmol TD₅₀, pmol PI Formalin Test (phase 2) CGX-1160 1 10-20 10-20CGX-1063 10-20 10-20 1-2 NT 600-700 3000   5-4.3 CFA-Induced AllodyniaTest CGX-1160 <0.1 10-20 >100 CGX-1063 <10 10-20 1-5 NT ≈500-600   30005-6 ED₅₀, pmol, estimated from antinociceptive tests TD₅₀, pmol,estimated from rotorod test of minimal motor impairment ProtectiveIndex, PI = (TD₅₀/ED₅₀)

Example 12 Materials and Methods for Assessing Antipsychotic Activity ofContulakin-G

1. Materials. D-amphetamine was obtained from Sigma (St. Louis, Mo.).Contulakin-G (CGX-1160; a synthetic 16 amino acid O-linked glycopeptide)was synthesized as described above.

2. Animals. Male CF-1 mice (30-35 g; Charles River Laboratories) wereused. All animals were housed in a temperature controlled (23°±3° C.)room with a 12 hour light-dark cycle with free access to food and water.All animals were euthanized in accordance with Public Health Servicepolicies on the humane care of laboratory animals.

3. Locomotor Activity. Animals were placed in clear plastic cages(40cm×22 cm, 20 cm deep) and allowed to acclimate for 30 minutes.Animals then received either contulakin-G (100 pmol) or saline (vehicle)by freehand intracerebroventricular (i.c.v.) injection (5 μl volume)through a 10 μl Hamilton syringe. After 5 minutes, animals receivedsaline or D-amphetamine sulphate (3 mg/kg) via intraperitoneal (i.p.)administration. Distance traveled (cm) and time spent ambulatory (s)were monitored for 30 minutes using a Videomex-V tracking system(Columbus Instruments, Columbus, Ohio). All testing was done in anisolated, dimly lit behavioral room.

4. Statistics. Data were analyzed using one-way analysis of variance(ANOVA) with drug treatment as the only factor, followed by aNewman-Keuls multiple comparison test for comparison of individualgroups, with P<0.05 accepted as statistically significant. Statisticalanalyses were performed with GraphPad PRISM software (Version 2.01,GraphPad, San Diego, Calif.).

Example 13 Antipsychotic Activity of Contulakin-G

A significant effect of drug treatment on locomotor activity as measuredby both distance traveled [F(4,21)=7.87, P<0.05] and time spentambulating [F(4,21)=6.17, P<0.05] was found in the present study.Administration of D-amphetamine resulted in a dose dependent increase inboth distance traveled and time spent ambulating (FIGS. 17-18).Pretreatment of mice with contulakin-G (100 pmol i.c.v.) significantlyreduced amphetamine-stimulated (3 mg/kg i.p.) increases in distancetraveled and time spent ambulating. A reduction in basal locomotoractivity (both distance traveled and time spent ambulating) was seenafter pretreatment with contulakin-G (100 pmol i.c.v.), however, thisreduction did not reach statistical significance.

Converging lines of evidence imply that neurotensin may haveantipsychotic properties without the associated adverse side effectprofiles of standard neuroleptic drugs (reviewed in (Nemeroff et al.,1992)). Subsequently, many groups have focused on neurotensin analogs asnovel antipsychotic drugs. Since contulakin-G shares C-terminal homologywith neurotensin, and resembles neurotensin in both in vivo and in vitroassays, the ability of contulakin-G to inhibit D-amphetamine-stimulatedlocomotor activity, a preclinical screen predictive of antipsychoticefficacy, was assessed. This example demonstrates that pretreatment ofmice with contulakin-G significantly reduced amphetamine-stimulatedincreases in locomotor activity. These data indicate that contulakin-Ghas similar antipsychotic activity as neurotensin. However as shownabove, while neurotensin was far more potent than contulakin-G at therat neurotensin receptors rNTR1 (IC₅₀: 3.2 nM for neurotensin; 524 nMfor contulakin-G) and rNTR2 (IC₅₀: 6.0 nM for neurotensin; 730 nM forcontulakin-G), and the mouse neurotensin receptor mNTR3 (IC₅₀: 1.4 nMfor neurotensin; 250 nM for contulakin-G), contulakin-G was 1 to 2orders of magnitude more potent in an in vivo assay (a visually ratedassessment of locomotor activity) following i.c.v. administration. Theseresults indicate that contulakin-G and neurotensin may interact withoverlapping but distinct populations of neurotensin receptor subtypes oractivation states. Thus, contulakin-G would not share the limiting sideeffects of neurotensin.

Example 14 Materials and Methods for Assessing Anticonvulsant Activityof Contulakin-G

1. Animals. Male Frings (20-25 g) were housed in a temperaturecontrolled (23°±1° C.) room with a 12 hour light-dark cycle with freeaccess to food and water. Mice were housed, fed, and handled in a mannerconsistent with the recommendations in HEW publication (NIH) No. 8623,“Guide for the Care and Use of Laboratory Animals.” All mice wereeuthanized in accordance with Public Health Service policies on thehumane care of laboratory animals.

2. Anticonvulsant Assessment. Frings mice were placed in a round,plexiglass jar (diameter 15 cm, height 18 cm) and exposed to a soundstimulus of 110 decibels (11 KHz). Mice were then observed for 25 secfor the presence or absence of hindlimb tonic extension. Animals notdisplaying hindlimb tonic extension were considered protected.

3. Rotorod Test. Motor impairment was assessed at time of peak effect byplacing mice on a rotorod turning at 6 rpm. Animals falling three timesin one minute were considered impaired.

Example 15 Anticonvulsant Activity of Contulakin-G

Contulakin-G (CGX-1160) and Thr₁₀-contulakin-G (CGX-1063) potently anddose-dependently blocked audiogenic seizures in Frings mice followingi.c.v. administration (FIG. 19). Similar to the efficacy in pain models,CGX-1160 was more potent than CGX-1063 with ED₅₀s of 7.1 pmol and 27.0pmol, respectively (Table 7). Also consistent with previous studies, NTwas dramatically less potent than CGX-1160 or -1063. Although adose-response curve for NT has not yet been completed, NT showed 50%protection following 1 nmol administered i.c.v. When tested for motortoxicity, CGX-1160 did not reach the 50% toxic level at doses up to 200pmol (FIG. 19), whereas the TD₅₀ for CGX-1063 is estimated to beapproximately 375 pmol resulting in an estimated PI of 14 for the dosestested.

TABLE 7 Anticonvulsant Profile of CGX-1160 and CGX-1063 in Frings AGSMice Following i.c.v. Administration X more Time of test TD₅₀ ED₅₀potent Compound (min.)^(a) (pmol) (pmol) P.I.^(b) than NT CGX-1160 15,60 >200 7.1 >28 ≈140 (4.9-8.5) CGX-1063 15, 60 ≈375 27.0  ≈14  ≈37(18.6-34.9) Neurotensin 15, 60 not yet ≈1000 N.D. determined ^(a)Firsttime, TD₅₀; second time, ED₅₀ ^(b)Protective index = TD₅₀/ED₅₀ ( ) 95%confidence interval

In a separate experiment, the time to peak effect and duration of actionof CGX-1160 was examined. I.c.v. administration of 100 pmol(approximately 14X ED₅₀) of CGX-1160 showed no activity at 30 minutes,but was 100% protective at 60 minutes, and still showed 50% protectionin animals tested 4 hours following i.c.v. injection (FIG. 20).

It will be appreciated that the methods and compositions of the instantinvention can be incorporated in the form of a variety of embodiments,only a few of which are disclosed herein. It will be apparent to theartisan that other embodiments exist and do not depart from the spiritof the invention. Thus, embodiments described are illustrative andshould not be construed as restrictive.

LIST OF REFERENCES

Annals of Pharmacotherapy 27:912 (1993).

Araki, K. et al. (1973). Chem. Pharm. Bull. (Tokyo) 21:2801-2804.

Barber, M. et al. (1982). Anal. Chem. 54:645A-657A.

Benziger, J. U. (1994). Faseb. J. 8:1019-1025.

Cancer 41:1270 (1993).

Cancer Res. 44:1698 (1984).

Carraway, R. et al. (1973). J. Biol. Chem. 248:6854-6861.

Cartier, G. E. et al. (1996). J. Biol. Chem. 271:7522-7528.

Chabry, J. et al. (1994). J. Neurochem. 63:19-27.

Chaplan, S. R. et al. (1994). J. Neurosci. Methods 53:55-63.

Clineschmidt, B. V. et al. (1979). Eur. J. Pharmacol. 54:129-139.

Colledge, C. J. et al. (1992). Toxicon.30:1111-1116.

Cotter, R. J. (1989). Biomed. Mass Spectrom. 18:513-532.

Craig, A. G. et al. (1993). Biol. Mass Spectrom. 22:31-44.

Craig, A. G. et al. (1994). Biol. Mass Spectrom. 23:519-528.

Craig, A. G. et al. (1998). Biochemistry 37:16019-16025.

Cruz, L. J. et al. (1987). Conus geographus toxins that discriminatebetween neuronal and muscle sodium channels. J. Biol. Chem.260:9280-9288.

Cusack, B. et al. (1993). J. Recept. Res. 13:123-134.

Cusack, B. et al. (1991). Eur. J. Pharmacol. 206:339-342.

Feurle, G. E. et al. (1992). J. Biol. Chem. 267:22305-22309.

Fischer, W. et al. (1987). Proc. Nat. Acad. Sci. USA 84:3628-3632.

Haack, J. A. et al. (1990). Contryphan-T: a gamma-carboxyglutamatecontaining peptide with N-methyl-d-aspartate antagonist activity. J.Biol. Chem. 265:6025-6029.

Hammerland, L. G. et al. (1992). Eur. J. Pharmacol. 226:239-244.

Hillenkamp, F. et al. (1993). Anal. Chem. 63:1193A-1203A.

Horiki, K. et al. (1978). Chemistry Letters 165-68.

Hylden, J. L. K. et al. (1980). Eur. J. Pharmacol. 67:313-316.

Jimenez, E. C. et al. (1996). J. Biol. Chem. 271:28002-28005.

Kaiser et al. (1970). Anal. Biochem. 34:595.

Kapoor (1970). J. Pharm. Sci. 59:1-27.

LeNguyen, D. and Rivier, J. (1986). Intl. J. Pep. Prot. 27:285-292.

Luning, B. et al. (1989). Glycoconjugate J. 6:5-19.

Malmberg, A. B et al. (1998). Pain 76:215-222.

Mazella, J. et al. (1988). J. Biol. Chem. 263:144-149.

Mcluckey, S. A. et al. (1991). Anal. Chem. 63:375-383.

Mena, E. E. et al. (1990). Contryphan-G: a novel peptide antagonist tothe N-methyl-D-aspartic acid (NMDA) receptor. Neurosci. Lett.118:241-244.

Methoden der Organischen Chemie (Houben-Weyl). Synthese von Peptiden, E.Wunsch (Ed.), Georg Thieme Verlag, Stuttgart, Ger. (1974).

Minamino, N. et al. (1984). Biochem. Biophys. Res. Commun. 122:542-549.

Monje, V. D. et al. (1993). Neuropharmacology 32:1141-1149.

Munson, P. J. et al. (1980). Anal. Biochem. 107:220-239.

Nishiuchi, Y. et al. (1993). Synthesis of gamma-carboxyglutamicacid-containing peptides by the Boc strategy. Int. J. Pept. Protein Res.42:533-538.

Nemeroff, C. B. et al. (1992). Ann. N.Y. Acad. Sci. 668:146-156.

Norberg, T. et al. (1994). In: Methods in Enzymology, Y. C. Lee Eds.,Academic Press, New York, N.Y., pp. 87-107.

Olivera, B. M. et al. (1984). Biochemistry 23:5087-5090.

Olivera, B. M. et al. (1985). Science 230:1338-1343.

Olivera, B. M. et al. (1990). Science 249:257-263.

Olivera, B. M. et al. (1997). Mol. Biol Cell 8:2101-2109.

Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co.,Easton, Pa. (1990).

Rivier, J. R. et al. (1978). Biopolymers 17:1927-38.

Rivier, J. R. et al. (1987). Biochem. 26:8508-8512.

Sadoul, J. L. et al. (1984). Biochem. Biophys. Res. Commun. 120:812-819.

Sambrook, J. et al. (1989). Molecular Cloning. A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

Schroder et al. (1965). The Peptides 1:72-75, Academic Press, New York.

Spengler, B. et al. (1992). Rapid Commun. Mass Spectrom. 6:105-108.

Stewart, J. M. et al. (1984). In: Pierce Chemical Company, Rockford,IL., pp 176.

Stewart, J. M. et al., Solid-Phase Peptide Synthesis, Freeman & Co., SanFrancisco, Calif. (1969).

Tanaka, K. et al. (1990). Neuron 4, 847-854.

Toth, I. et al. (1999). J. Med. Chem. (JMC ASAP web edition 22 September1999).

Van Renterghem, C. et al. (1988). Biochem. Biophys. Res. Comm.157:977-985.

Yoshida, H. et al. (1976). Biochemistry 15:61-64.

Zhou L. M., et al. (1996). Synthetic Analogues of Contryphan-G: NMDAAntagonists Acting Through a Novel Polyamine-Coupled Site. J. Neurochem.66:620-628.

U.S. Pat. No. 3,972,859.

U.S. Pat. No. 3,842,067.

U.S. Pat. No. 3,862,925.

U.S. Pat. No. 4,105,603.

U.S. Pat. No. 4,352,883.

U.S. Pat. No. 4,353,888.

U.S. Pat. No. 4,447,356.

U.S. Pat. No. 4,569,967.

U.S. Pat. No. 4,883,666.

U.S. Pat. No. 4,968,733.

U.S. Pat. No. 4,976,859.

U.S. Pat. No. 5,082,670.

U.S. Pat. No. 5,084,350.

U.S. Pat. No. 5,158,881.

U.S. Pat. No. 5,284,761.

U.S. Pat. No. 5,364,769.

U.S. Pat. No. 5,514,774.

U.S. Pat. No. 5,534,615.

U.S. Pat. No. 5,545,723.

U.S. Pat. No. 5,550,050.

U.S. Pat. No. 5,591,821.

U.S. Pat. No. 5,618,531.

U.S. Pat. No. 5,633,347.

U.S. application Ser. No. 08/785,534.

U.S. application Ser. No. 09/061,026.

PCT Published Application WO 92/19195.

PCT Published Application WO 94/25503.

PCT Published Application WO 95/01203.

PCT Published Application WO 95/05452.

PCT Published Application WO 96/02286.

PCT Published Application WO 96/02646.

PCT Published Application WO 96/11698.

PCT Published Application WO 96/40871.

PCT Published Application WO 96/40959.

PCT Published Application WO 97/12635.

13 1 16 PRT Conus geographus PEPTIDE (1)..(13) Xaa at residue 1 ispyro-Glu; Xaa at residue 13 is Pro or hydroxy-Pro; Thr at residue 10 ismodified to contain an O-glycan. 1 Xaa Ser Glu Glu Gly Gly Ser Asn AlaThr Lys Lys Xaa Tyr Ile Leu 1 5 10 15 2 16 PRT Artificial SequenceDescription of Artificial SequenceGeneric Contulakin-G formula 2 Xaa XaaXaa Xaa Gly Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ile Leu 1 5 10 15 3 17DNA Conus geographus misc_feature (1)..(17) n is any nucleotide 3atratnggyt tyttngt 17 4 15 PRT Conus geographus PEPTIDE (9) Xaa atresidue 9 is unknown 4 Ser Glu Glu Gly Gly Ser Asn Ala Xaa Lys Lys ProTyr Ile Leu 1 5 10 15 5 231 DNA Conus geographus CDS (1)..(228) 5 atgcag acg gcc tac tgg gtg atg gtg atg atg atg gtg tgg att gca 48 Met GlnThr Ala Tyr Trp Val Met Val Met Met Met Val Trp Ile Ala 1 5 10 15 gcccct ctg tct gaa ggt ggt aaa ctg aac gat gta att cgg ggt ttg 96 Ala ProLeu Ser Glu Gly Gly Lys Leu Asn Asp Val Ile Arg Gly Leu 20 25 30 gtg ccagac gac ata acc cca cag ctc atg ttg gga agt ctg att tcc 144 Val Pro AspAsp Ile Thr Pro Gln Leu Met Leu Gly Ser Leu Ile Ser 35 40 45 cgt cgt caatcg gaa gag ggt ggt tca aat gca acc aag aaa ccc tat 192 Arg Arg Gln SerGlu Glu Gly Gly Ser Asn Ala Thr Lys Lys Pro Tyr 50 55 60 att cta agg gccagc gac cag gtt gca tct ggg cca tag 231 Ile Leu Arg Ala Ser Asp Gln ValAla Ser Gly Pro 65 70 75 6 76 PRT Conus geographus 6 Met Gln Thr Ala TyrTrp Val Met Val Met Met Met Val Trp Ile Ala 1 5 10 15 Ala Pro Leu SerGlu Gly Gly Lys Leu Asn Asp Val Ile Arg Gly Leu 20 25 30 Val Pro Asp AspIle Thr Pro Gln Leu Met Leu Gly Ser Leu Ile Ser 35 40 45 Arg Arg Gln SerGlu Glu Gly Gly Ser Asn Ala Thr Lys Lys Pro Tyr 50 55 60 Ile Leu Arg AlaSer Asp Gln Val Ala Ser Gly Pro 65 70 75 7 16 PRT Conus geographusPEPTIDE (1)..(10) Xaa at residue 1 is pyro-Glu; Thr at residue 10contains an O-glycan. 7 Xaa Ser Glu Glu Gly Gly Glu Asn Ala Thr Lys LysPro Tyr Ile Leu 1 5 10 15 8 13 PRT Bos sp. PEPTIDE (1) Xaa at residue 1is pyro-Glu. 8 Xaa Leu Tyr Glu Asn Lys Pro Arg Arg Pro Tyr Ile Leu 1 510 9 6 PRT porcine 9 Lys Ile Pro Tyr Ile Leu 1 5 10 8 PRT Xenopus laevis10 Gln Gly Lys Arg Pro Trp Ile Leu 1 5 11 25 PRT Homo sapiens 11 Met LeuThr Lys Phe Glu Thr Lys Ser Ala Arg Val Lys Gly Leu Ser 1 5 10 15 PheHis Pro Lys Arg Pro Trp Ile Leu 20 25 12 17 PRT Vespula maculifrons 12Thr Ala Thr Thr Arg Arg Arg Gly Arg Pro Pro Gly Phe Ser Pro Phe 1 5 1015 Arg 13 9 PRT Homo sapiens 13 Arg Pro Pro Gly Phe Ser Pro Phe Arg 1 5

What is claimed is:
 1. A method for treating pain in an individual whichcomprises administering a therapeutically effective amount of an activeagent to a individual in need of pain treatment, said active agentselected from the group consisting of: (a) contulakin-G comprising theamino acid sequenceXaa₁-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Xaa₂-Tyr-Ile-Leu (SEQ IDNO:1), where Xaa₁ is pyro-Glu, Xaa₂ is proline or hydroxyproline andThr₁₀ is modified to contain O-glycan; b) a generic contulakin-G havingthe following general formulaXaa₁-Xaa₂-Xaa₃-Xaa₃-Gly-Gly-Xaa₂-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₇-Xaa₉-Xaa₁₀-lle-Leu(SEQ ID NO:2), where Xaa₁ is pyro-Glu, Glu, Gln or γ-carboxy-Glu; Xaa₂is Ser, Thr or S-glycan modified Cys; Xaa₃ is Glu or γ-carboxy-Glu; Xaa₄is Asn, N-glycan modified Asn or S-glycan modified Cys; Xaa₅ is Ala orGly; Xaa₆ is Thr, Ser, S-glycan modified Cys, Tyr or any hydroxycontaining unnatural amino acid; Xaa₇ is Lys, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine, homoarginine orany unnatural basic amino acid; Xaa₈ is Ala, Gly, Lys, N-methyl-Lys,N,N-dimethyl-Lys, N,N,N-trimethyl-Lys, Arg, ornithine, homoarginine, anyunnatural basic amino acid or X-Lys where X is (CH₂)_(n), phenyl,—(CH₂)_(m)—(CH═CH)—(CH₂)_(m)H or —(CH₂)_(m)—(C≡C)—(CH₂)_(m)H in which nis 1-4 and m is 0-2; Xaa₉ is Pro or hydroxy-Pro; and Xaa₁₀ is Tyr,mono-iodo-Tyr, di-iodo-Tyr, O-sulpho-Tyr, O-phospho-Tyr, nitro-Tyr, Trp,D-Trp, bromo-Trp, bromo-D-Trp, chloro-Trp, chloro-D-Trp, Phe, L-neo-Trp,or any unnatural aromatic amino acid, with the proviso that the genericcontulakin-G is not desglycosylated contulakin-G; (c) a genericcontulakin-G of (b) which is modified to contain an O-glycan, anS-glycan or an N-glycan; (d) a contulakin-G analog which comprises anN-terminal truncation of from 1 to 9 amino acids of the genericcontulakin-G of (b); (e) a contulakin-G analog of (c), wherein anSer-O-glycan, Thr-O-glycan or Cys-S-glycan is substituted for the aminoacid residue at the truncated N-terminus; (f) a contulakin-G analog of(c), wherein an Ser-O-glycan, Thr-O-glycan or Cys-S-glycan issubstituted for a residue at positions 2-9 of the generic contulakin-G;and (g) a contulakin-G analog which comprises an N-terminal truncationof 10 amino acids of the generic contulakin-G of (b) which is furthermodified to contain a Lys-N-glycan at residue 11 of the genericcontulakin-G.
 2. The method of claim 1, wherein said pain is acute pain.3. The method of claim 2, wherein said acute pain is post-trauma.
 4. Themethod of claim 1, wherein said pain is chronic pain.
 5. The method ofclaim 4, wherein said chronic pain results from cancer.
 6. The method ofclaim 4, wherein said chronic pain is neuropathic pain.
 7. The method ofclaim 4, wherein said chronic pain is inflammatory.
 8. The method ofclaim 1, wherein the active agent is administered using a deliverysystem selected from the group consisting of infusion, pump delivery,bioerodable polymer delivery, microencapsulated cell delivery, injectionand macroencapsulated cell delivery.
 9. The method of claim 8, whereinadministration is into the central nervous system.
 10. The method ofclaim 9, wherein the central nervous system is selected from the groupconsisting of the intrathecal space, the brain ventricles and the brainparenchyma.
 11. The method of claim 8, wherein the administration isselected from the group consisting of subcutaneous, intravenous,intra-arterial and intramuscular.